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A.C. Browning, W.M. Amoaku; The Isolation and Purification of Adult Human Sub-Macular Choroidal Endothelial Cells Using Anti-CD31 Coated Dynabeads . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3071.
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Purpose: To develop a method for the rapid isolation of human sub-macular choroidal endothelial cells. These could subsequently be cultured and used in experiments to further our understanding of choroidal neovascularisation. Method: Vitreous and retina were dissected from fresh human posterior segments. After scraping off of the RPE cells, a 8mm biopsy punch was used, centered on the fovea, to isolate the sub macular choroid. This was gently removed, cut into 1-2mm pieces and washed in isolation medium. The choroidal fragments were incubated in Dispase II 2.4U/ml and collagenase (0.5mg.ml) for 30 mins at 37°C with intermittent mixing. The homogenate was then filtered through a 40µM filter and the eluate washed in culture medium. The single cell suspension was mixed with 25µL of anti-CD31 coated Dynabeads (Dynal, Oslo,Norway) and incubated for 15 minutes at 4°C. The beads were washed in PBS and transferred to gelatin coated plates for culture in Endothelial growth medium (Sigma, Poole, UK). The cultured cells were tested for surface factor VIII by indirect immunohistochemistry. Results: The technique isolated numerous cells that were rosetted with anti-CD31 coated Dynabeads. These cells were positive for surface factor VIII, confirming them to be endothelial cells. Conclusions: We describe a method for rapid isolation of sub-macular choroidal endothelial cells, which can subsequently be utilised in the investigation of choroidal neovascularisation.
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