Coculture of ARPE-19 Cells and Porcine Neural Retina as an Ex Vivo Retinal Model.

Neural retinal organ cultures are used to investigate ocular pathomechanisms. However, these cultures lack the essential retinal pigment epithelium (RPE) cells, which are part of the actual in vivo retina. To simulate a more realistic ex vivo model, porcine neural retina explants were cocultured with ARPE-19 cells (ARPE-19 group), which are derived from human RPE. To identify whether the entire cells or just the cell factors are necessary, in a second experimental group, porcine neural retina explants were cultured with medium derived from ARPE-19 cells (medium group). Individually cultured neural retina explants served as controls (control group). After 8 days, all neural retinas were analysed to evaluate retinal thickness, photoreceptors, microglia, complement factors and synapses (n = 6-8 per group). The neural retina thickness in the ARPE-19 group was significantly better preserved than in the control group (p = 0.031). Also, the number of L-cones was higher in the ARPE-19 group, as compared to the control group (p < 0.001). Furthermore, the ARPE-19 group displayed an increased presynaptic glutamate uptake (determined via vGluT1 labelling) and enhanced post-synaptic density (determined via PSD-95 labelling). Combined Iba1 and iNOS detection revealed only minor effects of ARPE-19 cells on microglial activity, with a slight downregulation of total microglia activity apparent in the medium group. Likewise, only minor beneficial effects on photoreceptors and synaptic structure were found in the medium group. This novel system offers the opportunity to investigate interactions between the neural retina and RPE cells, and suggests that the inclusion of a RPE feeder layer has beneficial effects on the ex vivo maintenance of neural retina. By modifying the culture conditions, this coculture model allows a better understanding of photoreceptor death and photoreceptor-RPE cell interactions in retinal diseases.

Novel Porcine Retina Cultivation Techniques Provide Improved Photoreceptor Preservation.

Age-related macular degeneration (AMD) is the leading cause of blindness in industrialized countries among people over 60 years. It has multiple triggers and risk factors, but despite intense research efforts, its pathomechanisms are currently not completely understood. AMD pathogenesis is characterized by soft drusen in Bruch's membrane and involves the retinal pigment epithelium-Bruch's membrane-choroid complex and adjacent structures, like photoreceptors. This study explores the potential of novel cultivation techniques to preserve photoreceptors in retinal explants to gain better insights in AMD pathology. The porcine retina explants were cultured for 4 and 8 days using three different explantation techniques, namely, control (photoreceptors facing down, touching the filter), filter (photoreceptors facing up, turned sample using a filter), and tweezers (photoreceptors facing up, turned sample using tweezers). Optical coherence tomography revealed that the tweezers method had the best capacity to limit thinning of the retinal explants. Both novel methods displayed advantages in maintaining outer segment thickness. Additionally, immunofluorescence evaluation revealed a better preservation of opsin+ cells and rhodopsin signal intensity in both novel methods, especially the tweezers method. Furthermore, RT-qPCR analysis demonstrated an upregulation of OPSIN and RHODOPSIN mRNA expression in tweezers samples at 8 days. Amacrine and bipolar cell numbers were not altered at day 4 of cultivation, while cultivation until 8 days led to reduced bipolar cell numbers. At 4 days, CALRETININ mRNA was upregulated in filter samples, but protein kinase C alpha expression was downregulated. Retinal ganglion cells were diminished in both novel techniques due to a direct physical contact with the insert. Remarkably, no difference in TUBB3 mRNA expression was detected among the techniques. Nevertheless, both novel methods exhibited an improved retention of photoreceptor cells. In conclusion, the tweezers technique was the most promising one. Due to the high homology of the porcine to the human retina, it provides a reasonable alternative to in vivo rodent models. Consequently, an adapted coculture system based on the current findings may serve as an ex vivo model suitable to analyze AMD pathomechanisms and novel therapeutic approaches.