Abstract
Purpose :
The increasing need to develop alternative models to in vivo research results in different retinal organ culture models derived from mouse, rat, pig, primate, or human tissue. To mimic an in vivo-like environment, especially for photoreceptors and diseases of the outer retina like AMD, it is essential to co-cultivate retinas with RPE cells. Co-cultivation may be performed with primary-derived RPE cells or cell lines such as ARPE-19. Interestingly, indirect cultivation with RPE cells in porcine retinal cultures leads to signs of inflammation, although better photoreceptor survival was shown. To further investigate these effects, different passages of RPE cells derived from different pig eyes (allogenic), RPE cells derived from the same pig eye (autologous), and the ARPE-19 cell line were directly co-cultivated with porcine retinas.
Methods :
To confirm a functional monolayer of RPE cells, TEER measurements and ZO-1-stainings were performed. TUNEL-, calcein/PI-, cell viability-, and caspase assays as well as qRT-PCR with Bax/Bcl-2, RIPK3, and RIPK1 were performed to assess the cell death rate. Signs of inflammation were analyzed with qRT-PCR for IL-1β, IL-6, NLRP3, CD11b, HIF-1α, IL-8 markers, cytokine assays, and IBA1-staining. Lipid accumulation was visualized via Lipogreen2.
Results :
Functional RPE monolayers were generated with allogenic primary porcine RPE cells of passages 1-6. However, direct co-cultivation of RPE-P1 monolayers lead to inflammation and cell death in retinal explants and RPE cells. Interestingly, in RPE-P1 monolayers, direct co-cultivation also resulted in the accumulation of lipids in the degenerated RPE cells, indicating the development of drusen. Co-cultivation of retinal explants with autologous RPE cells did not induce proinflammatory genes. The same applies to co-cultivation with allogeneic RPE cells cultivated for high passages. Likewise, inflammation was also not caused by co-cultivation with ARPE-19 cells. In all different experimental setups, lower inflammation always resulted in a better photoreceptor condition.
Conclusions :
Allogenic RPE cells of higher passages did not induce apoptosis/inflammation to a critical amount. Therefore higher passages are needed for an ex vivo model without inflammation and for beneficial effects on retinal cells. In contrast, direct co-cultivation with primary allogeneic RPE monolayers from early passages could be used as an inflammatory AMD model.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.