Perfused 3D angiogenic sprouting in a high-throughput in vitro platform

Abstract

Angiogenic sprouting, the growth of new blood vessels from pre-existing vessels, is orchestrated by cues from within the cellular microenvironment, such as biochemical gradients and perfusion. However, many of these cues are missing in current in vitro models of angiogenic sprouting. We here describe an in vitro platform that integrates both perfusion and the generation of stable biomolecular gradients and demonstrate its potential to study more physiologically relevant angiogenic sprouting and microvascular stabilization. The platform consists of an array of 40 individually addressable microfluidic units that enable the culture of perfused microvessels against a three-dimensional collagen-1 matrix. Upon the introduction of a gradient of pro-angiogenic factors, the endothelial cells differentiated into tip cells that invaded the matrix. Continuous exposure resulted in continuous migration and the formation of lumen by stalk cells. A combination of vascular endothelial growth factor-165 (VEGF-165), phorbol 12-myristate 13-acetate (PMA), and sphingosine-1-phosphate (S1P) was the most optimal cocktail to trigger robust, directional angiogenesis with S1P being crucial for guidance and repetitive sprout formation. Prolonged exposure forces the angiogenic sprouts to anastomose through the collagen to the other channel. This resulted in remodeling of the angiogenic sprouts within the collagen: angiogenic sprouts that anastomosed with the other perfusion channel remained stable, while those who did not retracted and degraded. Furthermore, perfusion with 150 kDa FITC-Dextran revealed that while the angiogenic sprouts were initially leaky, once they fully crossed the collagen lane they became leak tight. This demonstrates that once anastomosis occurred, the sprouts matured and suggests that perfusion can act as an important survival and stabilization factor for the angiogenic microvessels. The robustness of this platform in combination with the possibility to include a more physiological relevant three-dimensional microenvironment makes our platform uniquely suited to study angiogenesis in vitro.

Keywords: 3D cell culture; Angiogenic sprouting; In vitro; Microfluidics; Vascular stabilization.

Fig. 1

Gradient generation in a 3D microenvironment. a Bottom of the OrganoPlate®, a microfluidic culture platform based on a 384-well plate. The glass bottom includes 40 microfluidic devices. b The geometry of a single microfluidic device that is positioned underneath nine wells (3 × 3). Every device consists of three channels: one ‘gel’ channel for gel patterning, and two adjacent channels. Phaseguides prevent the patterned gel from flowing into the adjacent channels. c Three-step method to generate gradients in patterned hydrogels. Step 1: 2 µL of collagen-1 gel is added in the center channel and polymerized. Step 2: source and sink are added in opposite perfusion channels. Step 3: the device is placed on a rocker platform to perfuse both perfusion channels continuously to generate a gradient. d Gradient visualization after 1, 3, and 6 days after addition of 20 kDa FITC-Dextran as a gradient source

Fig. 2

Microvessel culture against a  patterned collagen-1 gel. a Method the culture a microvessel within a microfluidic device. First, collagen-1 gel is patterned in the middle channel. After polymerization, an endothelial cell suspension was added in the adjacent perfusion channel. By placing the device on a rocker platform, the channels are continuously perfused. After 72 h, a confluent microvessel was formed. b Angiogenesis assay using a gradient of angiogenic factors. Angiogenic factors are added once a stable monolayer of ECs is formed against the gel (step 1). Addition of a gradient of angiogenic growth factors resulted in tip cells formation including filopodia at day 1 (step 2). Lumens formed by the stalk cells are visible at day 2 (step 3)

Fig. 3

Angiogenic sprouts after addition of angiogenic factors. a Images of sprouting after 4 days of stimulation of a gradient of different combinations of angiogenic factors. b Quantification of maximum absolute sprouting length in µm after stimulation for 3 (PMA) or 4 days (all other combinations) (n = 6). c Angiogenic sprouts after 6 days of stimulation with VEGF + PMA + S1P, stained against F-actin (red) and nucleus (blue). d Close-up of middle (i), top (ii), and cross-section (iii) of VEGF + PMA + S1P stimulated sprouting. Stained against F-actin (red) and nucleus (blue) and VE-cadherin (green). e Same as c, but stimulation with VEGF + PMA. f Same as c, but stimulation with VEGF + S1P. g–i Comparison between VEGF + PMA and VEGF + S1P in number of sprouts, diameter, and circularity (n = 2). Significance was calculated using one-way anova (b) or Student’s t test (g–i) and shown as n.s (non-significant), *(P < 0.05), **(P < 0.01), or ***(P < 0.001). Scale bars: 100 µm. Graphs are presented as mean ± SD

Fig. 4

Anastomosis with basal channel triggers pruning and maturation of angiogenic sprouts. a Angiogenic sprouts 5 days after addition of VEGF + PMA + S1P. Compared to the angiogenic sprouts at day 8. b Some sprouts regressed (arrows) while other sprouts remain and showed increased lumen diameter (arrowheads). c Angiogenic sprouts after 4 days of stimulation invaded into the gel but are not yet connected to the bottom perfusion channel. Fluorescent images were obtained every minute and directly after addition of a 0.5 mg/mL 150 kDa TRITC-Dextran solution in culture media. Panel ii shows the pseudo-colored fluorescent images after 0 and 9 min after addition of the dextran solutions. Time is indicated in min. d Same as in c, but after 9 days of stimulation. Sprouts are connected to the other side and formed a confluent microvessel in the basal perfusion channel. Scale bars: 100 µm