Isolation, culture, and characterisation of human macular inner choroidal microvascular endothelial cells

Abstract

Aim: To develop a method for the reliable isolation of adult human macular inner choroidal endothelial cells (ICECs) and to subsequently characterise them for their expression of a range of endothelial cell associated surface markers.

Method: Human ICECs were isolated after manual dissection of maculas from fresh human posterior segments. Following enzyme digestion to form a single cell suspension, the ICECs were isolated using anti-CD31 coated Dynabeads. The isolated cells were grown in culture and examined for typical endothelial cell morphology, surface expression of vWf, CD 31, CD 105, VEGF receptors 1 and 2, and expression of E-selectin after stimulation with TNF-alpha. The cells were also examined for their ability to form fenestrations and capillary-like tubes in Matrigel.

Results: The method enabled the rapid isolation of viable cells that demonstrated typical endothelial cobblestone morphology in culture. The cells stained positive for CD31, vWf, CD105, VEGF receptors 1 and 2, and E-selectin (after stimulation with TNF-alpha). The cells stained negative for alpha smooth muscle actin and fibroblast surface protein. The cells also developed fenestrations when cultured on fibronectin coated plates and formed capillary-like tubes structures when cultured on Matrigel.

Conclusions: This technique isolates cells from the human macular inner choroid that display features consistent with vascular endothelial cells. These cells could subsequently be used to further the understanding of the pathophysiological mechanisms of diseases of the inner choroid, such as choroidal neovascularisation.

Figure 1

Histological section of human submacular choroid stained with toludine blue (A) before and (B) after removal of the outer choroidal vessels. (B) Shows the residual choriocapillaris and inner choroidal vessels attached to Bruch’s membrane.

Figure 2

Phase contrast photomicrograph of confluent primary culture of ICECs demonstrating a typical cobblestone appearance (20× original magnification).

Figure 3

Immunofluorescent photomicrograph of human inner ICECs staining for (A) vWf, (B) CD31, (C) CD 105, (D) VEGF receptor 1, (E) VEGF receptor 2, (F) E-selectin (unstimulated), (G) E-selectin (post-TNF-α stimulation), (H)) mouse anti-rat isotype negative control, (I) α-SMA, (J) fibroblast surface protein, (K) human RPE cells stained for α-SMA, (L) human Tenon’s capsule fibroblasts stained for fibroblast surface protein. (All 63× original magnification.)

Figure 4

Transmission electron micrograph of human ICECs grown on a collagen IV coated Thermanox coverslip showing a typical fenestration (arrow) (bar = 200 nm).

Figure 5

Phase contrast photomicrograph of human ICECs 3 hours after seeding onto Matigel showing typical capillary-like tubes (20× original magnification).

Figure 6

Transmission electron micrograph of a lumen of a capillary-like tube surrounded by ICECs. The cells are joined by tight junctions (higher magnification in inset) and project villi into the lumen (bar = 2 μm).