研究目的
To develop an efficient protocol for the isolation and culture of primary mouse retinal pigment epithelial (RPE) cells that maintain in vivo characteristics, using inhibitors to prevent epithelial-mesenchymal transition.
研究成果
The optimized protocol efficiently isolates and cultures primary mouse RPE cells that maintain epithelial-like morphology and pigmentation, especially when treated with Y27632 and Repsox inhibitors. This method is reproducible and useful for studying RPE functions in genetic disease models, providing a reliable in vitro model that mimics in vivo conditions better than cell lines.
研究不足
Long-term in vitro culture leads to EMT in peripheral cells of RPE sheets, potentially limiting the duration for which cells maintain in vivo characteristics. The method may require careful handling to avoid contamination and ensure even cell distribution.
1：Experimental Design and Method Selection:
The study optimized a protocol for isolating and culturing primary RPE cells from C57BL mice, comparing cell sheet culture with single-cell culture, and using small molecule inhibitors (Y27632 and Repsox) to inhibit EMT. Theoretical basis includes previous methods and the role of Rho-kinase and TGFbR-1/ALK5 pathways in cell morphology.
2：Sample Selection and Data Sources:
C57BL/6 mice aged 7 days to 4 months were used as the source of RPE cells. No specific datasets were mentioned.
3：List of Experimental Equipment and Materials:
Equipment includes dissection stereomicroscope, Dumont #5 tweezers, Vannas scissors, p200 micropipette, laminar airflow hood, incubator (37°C, 5% CO2), centrifuge, and bright-field microscope. Materials include Dispase (2%, GIBCO), DMEM-F12 medium (GIBCO), fetal bovine serum (FBS, Hyclone), penicillin-streptomycin (P/S, GIBCO), Y27632 (50 μM, Sigma), Repsox (10 μM, Selleck), and various petri dishes and well plates.
4：Experimental Procedures and Operational Workflow:
Mice were sacrificed by cervical dislocation; eyes were enucleated and washed. RPE layer was separated using Dispase digestion (45 min at 37°C), incubated for 25 min to aid separation, scraped off choroid, collected, pipetted to reduce size, centrifuged, resuspended, and cultured in well plates with medium changes every 2 days. Inhibitors were added to treatment groups.
5：Data Analysis Methods:
Cell morphology was analyzed using bright-field microscopy to observe pigmentation, shape, and confluence. No statistical analysis or software tools were specified in the abstract.
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Dispase
GIBCO
Enzyme used for digesting and separating the RPE layer from the neural retina.
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DMEM-F12
GIBCO
Cell culture medium used for washing and growing RPE cells.
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Penicillin-Streptomycin
GIBCO
Antibiotic solution used to prevent bacterial contamination in cell culture.
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Fetal Bovine Serum
Hyclone
Serum supplement in cell culture medium to support cell growth.
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Y27632
Sigma
Rho-kinase inhibitor used to inhibit epithelial-mesenchymal transition in RPE cells.
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Repsox
Selleck
TGFbR-1/ALK5 inhibitor used to inhibit epithelial-mesenchymal transition in RPE cells.
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Dissection Stereomicroscope
Used for dissecting mouse eyes under sterile conditions.
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Dumont Tweezers
#5
Used for handling and fixing eye tissues during dissection.
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Vannas Scissors
8-cm
Used for cutting eye tissues during dissection.
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Micropipette
p200
Used for pipetting and transferring RPE cells and solutions.
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Incubator
Maintains cell culture at 37°C with 5% CO2.
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Centrifuge
Used for centrifuging RPE cells to pellet them.
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Bright-field Microscope
Used for observing cell morphology and confluence.
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