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Updated: Jun 17, 2025
Primary Culture of Porcine Retinal Pigment Epithelial Cells
Published on: September 23, 2022
Retinal debris triggers cytotoxic damage in cocultivated primary porcine RPE cells
Natalie Wagner1, Teresa Tsai1, Sabrina Reinehr1
1Experimental Eye Research Institute, University Eye Hospital, Ruhr-University Bochum, Bochum, Germany.
Introduction:
One of the most common causes of vision loss in the elderly population worldwide is age-related macular degeneration (AMD). Subsequently, the number of people affected by AMD is estimated to reach approximately 288 million by the year 2040. The aim of this study was to develop an ex vivo model that simulates various aspects of the complex AMD pathogenesis.
Methods:
For this purpose, primary porcine retinal pigment epithelial cells (ppRPE) were isolated and cultured. One group was exposed to medium containing sodium iodate (NaIO3) to induce degeneration. The others were exposed to different supplemented media, such as bovine serum albumin (BSA), homogenized porcine retinas (HPR), or rod outer segments (ROOS) for eight days to promote retinal deposits. Then, these ppRPE cells were cocultured with porcine neuroretina explants for another eight days. To assess the viability of ppRPE cells, live/dead assay was performed at the end of the study. The positive RPE65 and ZO1 area was evaluated by immunocytochemistry and the expression of RLBP1, RPE65, and TJP1 was analyzed by RT-qPCR. Additionally, drusen (APOE), inflammation (ITGAM, IL6, IL8, NLRP3, TNF), oxidative stress (NFE2L2, SOD1, SOD2), and hypoxia (HIF1A) markers were investigated. The concentration of the inflammatory cytokines IL-6 and IL-8 was determined in medium supernatants from day 16 and 24 via ELISA.
Results:
Live/dead assay suggests that especially exposure to NaIO3 and HPR induced damage to ppRPE cells, leading in a significant ppRPE cell loss. All supplemented media resulted in decreased RPE-characteristic markers (RPE65; ZO-1) and gene expression like RLBP1 and RPE65 in the cultured ppRPE cells. Besides, some inflammatory, oxidative as well as hypoxic stress markers were altered in ppRPE cells cultivated with NaIO3. The application of HPR induced an enhanced APOE expression. Pre-exposure of the ppRPE cells led to a diminished number of cones in all supplemented media groups compared to controls.
Discussion:
Overall, this novel coculture model represents an interesting initial approach to incorporating deposits into coculture to mimic AMD pathogenesis. Nevertheless, the effects of the media used need to be investigated in further studies.
Insights
This study developed an ex vivo model for age-related macular degeneration (AMD) using porcine cells. The model showed promise in mimicking AMD pathogenesis, with specific media inducing cell damage and altering key markers.
Area of Science:
- Ophthalmology
- Cell Biology
- Biomedical Engineering
Background:
- Age-related macular degeneration (AMD) is a leading cause of vision loss in the elderly, affecting millions globally.
- The projected increase in AMD cases necessitates advanced research models to study its complex pathogenesis.
- Current models often lack the complexity to fully replicate AMD's multifaceted nature.
Purpose of the Study:
- To develop a novel ex vivo coculture model simulating key aspects of AMD pathogenesis.
- To investigate the effects of specific media compositions on porcine retinal pigment epithelial (ppRPE) cells and neuroretina explants.
- To assess the utility of this model in studying cellular damage, deposit formation, and associated molecular markers relevant to AMD.
Main Methods:
- Primary porcine RPE cells (ppRPE) were cultured and exposed to sodium iodate (NaIO3) or supplemented media (BSA, HPR, ROOS) to induce degeneration and deposits.
- ppRPE cells were cocultured with porcine neuroretina explants for eight days.
- Cell viability, RPE-characteristic markers (RPE65, ZO-1), gene expression (RLBP1, RPE65, TJP1), and markers for drusen, inflammation, oxidative stress, and hypoxia were analyzed.
Main Results:
- Exposure to NaIO3 and homogenized porcine retinas (HPR) significantly damaged ppRPE cells, causing cell loss.
- Supplemented media reduced RPE-characteristic markers and gene expression.
- Inflammatory, oxidative, and hypoxic stress markers were altered, with HPR inducing increased APOE expression. Cone numbers diminished in supplemented groups.
Conclusions:
- The developed coculture model offers a promising initial approach for mimicking AMD pathogenesis, including deposit formation.
- The model's ability to induce cellular damage and alter relevant molecular pathways warrants further investigation.
- Additional studies are needed to fully elucidate the effects of different media components within this novel AMD model.
