Related Experiment Video
Updated: Oct 5, 2025
Isolation of Primary Mouse Retinal Glial Müller Cells
Published on: August 30, 2024
Isolation and Characterization of Primary Retinal Microglia From the Human Post-mortem Eyes for Future Studies of
Luoziyi Wang1, Yiwen Qian1, Xin Che1
1Department of Ophthalmology, Huashan Hospital, Fudan University, Shanghai, China.
Abstract:
Microglia, the primary resident immunocytes in the retina, continuously function as immune system supervisors in sustaining intraocular homeostasis. Microglia relate to many diseases, such as diabetic retinopathy, glaucoma, and optic nerve injury. To further investigate their morphology and functions in vitro, a reliable culture procedure of primary human retinal microglia is necessary. However, the culture condition of microglia from the adult retina is unclear. Researchers created several protocols, but most of them were carried out on rodents and newborns. This study describes a protocol to isolate and characterize human primary retinal microglia from human post-mortem eyes. The whole procedure started with removing the retinal vessels, mechanical separation and enzymatic dissociation, filtration, and centrifugation. Then, we cultured the cell suspensions on a T-75 flask for 18 days and then shook retinal microglia from other retinal cells. We found numerous retinal microglia grow and attach to Müller cells 10 days after seeding and increase rapidly on days 14-18. Iba1 and P2RY12 were used to qualify microglia through immunofluorescence. Moreover, CD11b and P2RY12 were positive in flow cytometry, which helps to discriminate microglia from other cells and macrophages. We also observed a robust response of retinal microglia in lipopolysaccharide (LPS) treatment with proinflammatory cytokines. In conclusion, this study provides an effective way to isolate and culture retinal microglia from adult human eyes, which may be critical for future functional investigations.
Insights
Researchers developed a new method to isolate and culture human retinal microglia from adult post-mortem eyes. This protocol is crucial for studying microglia function in various eye diseases.
Area of Science:
- Ophthalmology
- Immunology
- Cell Biology
Background:
- Microglia are vital retinal immune cells maintaining intraocular homeostasis.
- Retinal microglia dysfunction is linked to diseases like diabetic retinopathy, glaucoma, and optic nerve injury.
- Existing culture protocols often use rodent or newborn models, limiting human adult retinal microglia research.
Purpose of the Study:
- To establish a reliable protocol for isolating and characterizing primary human retinal microglia from adult post-mortem eyes.
- To enable in vitro studies of microglia morphology and function.
Main Methods:
- Isolation involved retinal vessel removal, mechanical and enzymatic dissociation, filtration, and centrifugation.
- Cells were cultured for 18 days, with microglia separated via shaking.
- Immunofluorescence (Iba1, P2RY12) and flow cytometry (CD11b, P2RY12) were used for characterization.
Main Results:
- Successful isolation and culture of primary human retinal microglia.
- Microglia attached to Müller cells around day 10, with rapid proliferation on days 14-18.
- Characterization confirmed microglia identity and demonstrated inflammatory response to lipopolysaccharide (LPS).
Conclusions:
- This study presents an effective protocol for isolating and culturing adult human retinal microglia.
- The method provides a valuable tool for future in vitro functional investigations of retinal microglia.
- This research may advance understanding and treatment of human retinal diseases.
