Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro
Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro
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DOI：
10.1096/fj.14-267633
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发表时间：
2015-07-01
期刊：
影响因子：
4.8
通讯作者：
Korff, Thomas
中科院分区：
文献类型：
--
作者：
Heiss, Maximilian;Hellstrom, Mats;Korff, Thomas
考虑到需要稳健且具有成本效益的体外模型来研究血管生成并在临床前研究中可重复地分析潜在的促血管生成和抗血管生成化合物，我们开发了一种三维体外血管生成测定法，该测定法基于从培养的人脐静脉内皮细胞（HUVEC）产生的胶原凝胶包埋的、大小确定的球状体。尽管其广泛的分布，局限性，灵敏度，鲁棒性，和改进，该试验的功能筛选目的的能力尚未阐明迄今为止。通过使用延时视频显微镜，我们表明，尖端细胞导致的毛细管样和部分管腔化的芽起源于球体的形成。血管内皮细胞生长因子165的生理浓度诱导5种不同的原代培养的人内皮细胞类型产生的球状体的血管生成出芽。基于该检测系统，我们确定了880种已批准药物干扰或促进血管生成发芽的能力，从而评估了其推定的血管生成相关副作用或新应用。然而，尽管该测定允许快速且可重现地测定单个化合物的功能性IC 50值，但发芽结果部分受到HUVEC传代次数和供体变异性的影响。为了克服这一限制，在本研究过程中使用了在多个群体倍增中增殖和发芽方面显示出更均一反应的永生化HUVEC（iHUVEC）。总的来说，基于球状体的血管生成测定提供了一种灵敏且通用的工具来研究促血管生成和抗血管生成决定因素对血管生成级联反应的多个步骤的影响。它与不同的内皮细胞类型相容，并允许使用iHUVEC来提高其整体稳健性。
Given the need for robust and cost-efficient in vitro models to study angiogenesis and reproducibly analyze potential pro-and antiangiogenic compounds in preclinical studies, we developed a 3-dimensional in vitro angiogenesis assay that is based on collagen gel-embedded, size-defined spheroids generated from cultured human umbilical vein endothelial cells (HUVECs). Despite its wide distribution, limitations, sensitivity, robustness, and improvements, the capacity of this assay for functional screening purposes has not been elucidated thus far. By using time-lapse video microscopy, we show that tip cells lead the formation of capillary-like and partially lumenized sprouts originating from the spheroids. Angiogenic sprouting from spheroids generated from 5 different primary cultured human endothelial cell types was induced by physiologic concentrations of vascular endothelial cell growth factor 165. Based on this assay system, we determined the capacity of 880 approved drugs to interfere with or boost angiogenic sprouting, thereby assessing their putative angiogenesis-related side effects or novel applications. However, although this assay allowed for a rapid and reproducible determination of functional IC50 values of individual compounds, the sprouting results were partially affected by the HUVEC passage number and donor variability. To overcome this limitation, immortalized HUVECs (iHUVECs) showing a more homogenous response in terms of proliferation and sprouting over multiple population doublings were used in the course of this study. Collectively, the spheroid-based angiogenesis assay provides a sensitive and versatile tool to study the impact of pro-and antiangiogenic determinants on multiple steps of the angiogenic cascade. It is compatible with different endothelial cell types and allows use of iHUVECs to improve its overall robustness.