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Updated: Nov 7, 2025
Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes
Published on: May 9, 2023
A modified protocol for isolation of retinal microglia from the pig
Yahan Zhang1, Anna Lena Erhard1, Tanja Plagemann1
1University of Münster Medical School, Department of Ophthalmology, Münster, Germany.
Abstract:
Microglia are the resident immune cells in the retina. To investigate their properties and behaviour, a reliable and yielding procedure to culture them is necessary. We here describe a way of isolation of microglial cells from the porcine retina, as pig eyes are similar to human eyes in size, structure and vasculature, including similarities in proteins and pathways. Retina was isolated from fresh pig eyes, dissociated by a mixture of collagenase, hyaluronidase and DNAse, and passed through a cell strainer. After triple centrifugation with decreasing velocity and re-suspension, cells were seeded into poly-d-lysine coated culture flasks and cultured using DMEM and macrophage-colony stimulating factor (M-CSF). Number of cells increased gradually during the first 10-14 days, till they could be split and used for experiments. Identity of isolated cells as microglia was assessed by immunostaining against the microglia/macrophage markers Iba1, CD11b, CD68, CD45 and TMEM119. Phagocytic function of microglia could be demonstrated by phagocytosis of fluorescence beads and their response to lipopolysaccharide (LPS). As a conclusion, we developed a protocol for isolation and cultivation of pig retinal microglial cells that are suitable for research in the laboratory.
Insights
Researchers developed a new protocol to isolate and culture pig retinal microglial cells. This method provides a reliable source of these immune cells for laboratory research.
Area of Science:
- Ophthalmology
- Immunology
- Cell Biology
Background:
- Microglia are essential resident immune cells in the retina.
- Understanding microglial function requires reliable in vitro culture methods.
- Porcine eyes offer anatomical and molecular similarities to human eyes.
Purpose of the Study:
- To establish a reproducible protocol for isolating and culturing porcine retinal microglial cells.
- To validate the identity and functionality of the cultured microglial cells.
Main Methods:
- Porcine retinas were dissociated using enzymatic treatment (collagenase, hyaluronidase, DNAse).
- Cells were cultured in DMEM supplemented with macrophage-colony stimulating factor (M-CSF) on poly-d-lysine coated flasks.
- Microglial identity was confirmed via immunostaining for specific markers (Iba1, CD11b, CD68, CD45, TMEM119).
- Phagocytic activity was assessed using fluorescent beads and response to lipopolysaccharide (LPS).
Main Results:
- A gradual increase in cell numbers was observed over 10-14 days, allowing for cell splitting.
- Immunostaining confirmed the presence of microglia/macrophage markers.
- Cultured cells demonstrated phagocytic capabilities and responded to LPS stimulation.
Conclusions:
- A robust protocol for isolating and cultivating retinal microglial cells from porcine eyes was successfully developed.
- These cultured porcine microglial cells are suitable for various laboratory research applications.
- This method provides a valuable model for studying retinal immune cell behavior.
