Huuskes Brooke M, DeBuque Ryan J, Kerr Peter G, Samuel Chrishan S, Ricardo Sharon D
Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Melbourne, VIC, Australia.
Australian Regenerative Medicine Institute, Monash University, Melbourne, VIC, Australia.
Front Cell Dev Biol. 2019 Apr 10;7:45. doi: 10.3389/fcell.2019.00045. eCollection 2019.
Testing angiogenic potential and function of cells in culture is important for the understanding of the mechanisms that can modulate angiogenesis, especially when discovering novel anti- or pro-angiogenic therapeutics. Commonly used angiogenic assays include tube formation, proliferation, migration, and wound healing, and although well-characterized, it is important that methodology is standardized and reproducible. Human endothelial progenitor cells (EPCs) are critical for post-natal vascular homeostasis and can be isolated from human peripheral blood. Endothelial colony forming cells (ECFCs) are a subset of EPCs and are of interest as a possible therapeutic target for hypoxic diseases such as kidney disease, as they have a high angiogenic potential. However, once ECFCs are identified in culture, the exact timing of passaging has not been well-described and the optimal conditions to perform angiogenic assays such as seeding density, growth media (GM) concentrations and end-points of these assays is widely varied in the literature. Here, we describe the process of isolating, culturing and passaging ECFCs from patients with end-stage renal disease (ESRD), aided by image analysis. We further describe optimal conditions, for human bladder endothelial cells (hBECs), challenged in angiogenic assays and confirm that cell density is a limiting factor in accurately detecting angiogenic parameters. Furthermore, we show that GM along is enough to alter the angiogenic potential of cells, seeded at the same density. Lastly, we report on the success of human ECFCs in angiogenic assays and describe the benefits of live-cell imaging combined with time-lapse microscopy for this type of investigation.
检测培养细胞的血管生成潜力和功能对于理解可调节血管生成的机制非常重要，特别是在发现新型抗血管生成或促血管生成疗法时。常用的血管生成检测方法包括管腔形成、增殖、迁移和伤口愈合，尽管这些方法已得到充分表征，但方法的标准化和可重复性很重要。人内皮祖细胞（EPCs）对出生后血管稳态至关重要，可从人外周血中分离出来。内皮集落形成细胞（ECFCs）是EPCs的一个子集，由于其具有高血管生成潜力，作为诸如肾脏疾病等缺氧疾病的可能治疗靶点而受到关注。然而，一旦在培养物中鉴定出ECFCs，传代的确切时间尚未得到很好的描述，并且进行血管生成检测的最佳条件，如接种密度、生长培养基（GM）浓度和这些检测的终点，在文献中差异很大。在这里，我们描述了在图像分析的辅助下，从终末期肾病（ESRD）患者中分离、培养和传代ECFCs的过程。我们进一步描述了人膀胱内皮细胞（hBECs）在血管生成检测中的最佳条件，并证实细胞密度是准确检测血管生成参数的限制因素。此外，我们表明仅GM就足以改变以相同密度接种的细胞的血管生成潜力。最后，我们报告了人ECFCs在血管生成检测中的成功，并描述了活细胞成像与延时显微镜相结合用于此类研究的好处。
