Endothelial cell spheroids as a versatile tool to study angiogenesisin vitro

Abstract

Given the need for robust and costefficient 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 IC 50 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.-Heiss, M

Decoupling the effects of stiffness and fiber density on cellular behaviors via an interpenetrating network of gelatin-methacrylate and collagen

“…Assessment of EC spheroid sprouting, a process that involves invasion in tandem with the development of vessel-like structures in the surrounding matrix, is often used as a means for evaluating angiogenic potential [41]. At 72 hours following embedding, viable cells within the spheroids were fluorescently stained with calcein AM (Figure 4A), and the total length of all EC sprouts emerging from each spheroid was quantified.…”

In Vitro Multitissue Interface Model Supports Rapid Vasculogenesis and Mechanistic Study of Vascularization across Tissue Compartments

“…Instead, vessels sprout and extend from the spheroid into the surrounding matrix, mimicking angiogenesis. 48 …”

miR‐191 suppresses angiogenesis by activation of NF‐kB signaling

“…HDMECs were suspended in EGM‐MV containing 0.24% (w/v) methylcellulose (Sigma‐Aldrich) and seeded (500 cells/100 μl) in nonadherent, round‐bottom, 96‐well plates (Greiner Bio‐One, Frickenhausen, Germany). After incubation for 24 h, spheroids were harvested and resuspended in collagen solution as previously described (24). The spheroid‐containing collagen gel (~150 spheroids/ml) was rapidly transferred to 24‐well plates (300 μl/well) and allowed to polymerize for 45 min, after which 0.4 ml EGM‐MV was added to each well.…”