SRS242 - Establishment and functional characterisation of a primary human renal vascular model <i>in vitro</i>

Abstract Background The endothelium is the primary site of recipient attack after transplantation. Endothelial cells (EC) are extremely susceptible to ischaemia-reperfusion injury, and EC dysfunction mediates inflammation, immune cell recruitment and complement activation. However, many in vitro endothelial models are derived from non-renal vessels, and establishing pure primary cell cultures from kidney biopsies is challenging. Here, we describe the establishment and functional characterisation of human renal vein endothelial cells (hRVECs), demonstrating a simple and high-yielding method of EC extraction for use as a vascular model in transplantation. Methods and Results Renal veins were dissected from n = 10 human kidneys declined for transplantation and offered for research. Following collagenase digestion and primary cell isolation, CD31+ (PECAM – a constitutively expressed EC marker) cells were purified at passage 1 (P1). Pure, healthy EC cultures were obtained in all vessel isolations, including where vessels had &amp;gt;44h cold ischaemic time. Cells were phenotyped between P1-P6, demonstrating &amp;gt;98% CD31+ and CD144+ cells. In-depth phenotyping also showed all tested hRVECs were negative for markers of cellular contamination (EpCAM and CD45). hRVECs were capable of forming capillary-like vessels in 3D culture in 2–6 h, and upregulated ICAM-1, VCAM-1, and HLA Class I and Class II molecules in response to cytokine stimulation. Conclusions With &amp;lt;1h hands-on time for primary cell extraction, and vessel storage possible for up to 44 h, we demonstrate a practical and simple method of endothelial cell isolation from multiple human kidney donors. hRVECs therefore present a promising and relevant primary cell model for renal transplantation research.