Isolation, culture, and characterisation of human macular inner choroidal microvascular endothelial cells
Isolation, culture, and characterisation of human macular inner choroidal microvascular endothelial cells
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DOI：
10.1136/bjo.2004.063602
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发表时间：
2005-10-01
影响因子：
4.1
通讯作者：
Amoaku, WM
中科院分区：
文献类型：
--
作者：
Browning, AC;Gray, T;Amoaku, WM
目的：为了开发一种方法，成人黄斑脉络膜内皮细胞（ICECs）的可靠分离，并随后将其表达的一系列内皮细胞相关的表面markets.Method：人ICECs分离后，手动解剖新鲜的人后段黄斑。在酶消化以形成单细胞悬浮液后，使用抗CD 31包被的Dynabeads分离ICEC。分离的细胞在培养物中生长，并检查典型的内皮细胞形态，vWf、CD 31、CD 105、VEGF受体1和2的表面表达，以及用TNF-α刺激后E-选择素的表达。细胞也进行了检查，他们的能力，形成开窗和毛细血管样管Matrigel.Results：该方法能够快速分离的活细胞，表现出典型的内皮鹅卵石形态的文化。细胞对CD 31、vWf、CD 105、VEGF受体1和2以及E-选择素（用TNF-α刺激后）染色呈阳性。细胞对平滑肌肌动蛋白和成纤维细胞表面蛋白染色呈阴性。细胞也开发开窗培养时，纤连蛋白包被的板和形成毛细血管样管结构时，培养Matrigel.Conclusions：这项技术分离的细胞从人黄斑内脉络膜显示功能与血管内皮细胞一致。这些细胞随后可用于进一步了解内部脉络膜疾病的病理生理机制，如脉络膜新生血管形成。
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 a 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.