Shishkova Daria, Yurieva Yulia, Frolov Alexey, Matveeva Vera, Torgunakova Evgenia, Markova Victoria, Lazebnaya Anastasia, Kutikhin Anton
Department of Experimental Medicine, Research Institute for Complex Issues of Cardiovascular Diseases, 6 Barbarash Boulevard, 650002 Kemerovo, Russia.
Int J Mol Sci. 2025 Sep 21;26(18):9217. doi: 10.3390/ijms26189217.
Primary human endothelial cells represent an essential tool to model endothelial dysfunction and to screen interventions for its treatment. Here, we developed a protocol for the synchronous isolation of primary human saphenous vein endothelial cells (HSaVEC), human internal thoracic artery endothelial cells (HITAEC), and human microvascular endothelial cells (HMVEC) from SV and ITA utilized as conduits during coronary artery bypass graft surgery and from subcutaneous adipose tissue excised while providing an access to the heart. Treatment by collagenase type IV and magnetic separation with anti-CD31-antibody-coated beads ensured relatively high efficiency of the isolation (≈60% for HSaVEC, ≈50% for HITAEC, and ≈20% for HMVEC) and high purity (≥99%) of isolated ECs within ≈2 weeks (HSaVEC), ≈2-3 weeks (HITAEC), and ≈3-4 weeks (HMVEC). A colorimetric assay of cell viability and proliferation, as well as real-time bioimpedance monitoring using the xCELLigence instrument, demonstrated high proliferative activity in HSaVEC, HITAEC, and HMVEC, whilst the in vitro tube formation assay indicated their angiogenic potential. The isolation of HSaVEC, HITAEC, and HMVEC from patients undergoing coronary artery bypass graft surgery is a promising option to investigate endothelial heterogeneity, to interrogate endothelial responses to various stresses, and to pinpoint the optimal approaches for restoring endothelial homeostasis, thereby reproducing them within the bedside-to-bench-to-bedside concept.
原代人内皮细胞是模拟内皮功能障碍和筛选其治疗干预措施的重要工具。在此，我们开发了一种方案，用于同步分离原代人隐静脉内皮细胞（HSaVEC）、人胸廓内动脉内皮细胞（HITAEC）和人微血管内皮细胞（HMVEC），这些细胞分别取自冠状动脉搭桥手术中用作血管 conduit 的隐静脉（SV）和胸廓内动脉（ITA），以及在提供心脏通路时切除的皮下脂肪组织。通过 IV 型胶原酶处理和用抗 CD31 抗体包被的磁珠进行磁分离，确保了相对较高的分离效率（HSaVEC 约为 60%，HITAEC 约为 50%，HMVEC 约为 20%），并且在约 2 周（HSaVEC）、约 2 - 3 周（HITAEC）和约 3 - 4 周（HMVEC）内，分离得到的内皮细胞纯度高（≥99%）。细胞活力和增殖的比色测定以及使用 xCELLigence 仪器进行的实时生物阻抗监测表明，HSaVEC、HITAEC 和 HMVEC 具有高增殖活性，而体外管形成试验表明它们具有血管生成潜力。从接受冠状动脉搭桥手术的患者中分离 HSaVEC、HITAEC 和 HMVEC 是一种有前景的选择，可用于研究内皮细胞异质性、探究内皮细胞对各种应激的反应以及确定恢复内皮稳态的最佳方法，从而在床边到实验台再到床边的概念中重现这些过程。
