Robust gradient formation in a 3D microenvironment
The microfluidic culture platform is based on a 384-well microtiter plate format. The glass bottom contains 40 microfluidic units (Fig. 1a), and each microfluidic unit is positioned underneath nine wells (3 × 3). Every unit consists of three channels: the center channel that is used to pattern an extracellular matrix (‘gel channel’) and two adjacent channels (‘perfusion channels’) (Fig. 1b). The channels are separated by PhaseGuides: small ridges that function as capillary pressure barriers, which enable patterning of cells and gel without the use of artificial membranes . Every channel has one inlet and one outlet, which connect the channels with the wells in the microtiter plate. Compartmentalization is achieved by patterning a hydrogel in the middle channel (Fig. 1c, step 1), and enables the formation of gradients by adding a source and sink in the opposite perfusion channels (Fig. 1c, step 2). Without continuous replenishment of the gradients source and sink in the microfluidic channels, gradients typically last only a few minutes (data not shown). To stabilize the gradient over time, the device was placed on a rocker platform to perfuse both perfusion channels continuously and simultaneously (Fig. 1c, step 3). As the volume inside the wells is typically orders of a magnitude higher than in the microfluidic channels (the wells typically contain volumes of 50 µL, compared to < 1 µL in the microfluidic channels), the source and sink within the microfluidic channels are constant over prolonged periods of time. Thus, a stable gradient could be maintained for multiple days (Fig. 1d) without the need to replenish. Although a gradient is still present after 6 days, the steepness is affected due to saturation of the sink. Therefore, growth factors and medium were replaced at 2–3-day time intervals.
Importantly, the high hydraulic resistance of the hydrogel limits the influence of differences in hydrostatic pressures. This results in a reproducible and robust platform to generate gradients, despite the presence of small difference in volumes, for example, due to pipetting errors. Nonetheless, hydrostatic pressures still can influence the shape of the gradient, when the difference between the volumes is sufficiently large. This allows different types of gradient to be generated (e.g., linear or parabolic, Supplementary Fig. 1).
Microvessels cultured against patterned collagen-1 gel
After gel loading and polymerization (Fig. 2a, step 1), endothelial cell suspensions were added to the perfusion channels adjacent to the gel. After the cells adhered to the glass substrate (step 2) of the channel, perfusion was applied by placing the device on a rocker platform (step 3). Confluent microvessels were formed after 3 days of culture, and the apical side of the vessel (the lumen) can be accessed through the perfusion channel, while the gel forms the basal side of the tube .
Combination of angiogenic factors is required to induce sprouting
After reaching confluency in 3 days, the microvessels showed a stable morphology of a single monolayer against the gel (Fig. 2b, step 1), despite the numerous (angiogenic) growth factors that are present in the media (such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)). We included VEGF and S1P as they have been shown to induce angiogenic sprouting within a collagen-1 matrix [39,40,41] and included phorbol 12-myristate 13-acetate (PMA) as it has been found to promote lumen formation in the absence of fibroblasts [15, 42], and used in concentrations of 50 ng/mL for VEGF, 500 nM for S1P, and 2 ng/mL for PMA. The angiogenic growth factor cocktail was added on the basal side of the vessels, and formed a gradient within the collagen-1 gel (Fig. 2b, step 1). This induced the formation of tip and stalk cells after respectively 1 and 2 days (Fig. 2b, step 2–3).
Interestingly, adding either VEGF, S1P, or PMA alone on the basal side did not result in angiogenic sprouting (Supplementary Fig. 2). We quantified the angiogenesis after addition of various combinations of VEGF, PMA and S1P (Fig. 3a, b). VEGF + PMA + S1P together resulted in angiogenesis including tip/stalk cell formation, the presence of filopodia and lumen formation and directional growth towards the gradient. The sprouts fully traversed the gel after about 6 days and started to form a continuous monolayer against in the channel on the other side of the gel and in the basal perfusion channel (Fig. 3c). The angiogenic sprouts have a clear lumen formation (Fig. 3d, panel i), appear circular in a cross-sectional view (Fig. 3d, panel ii), and have clear VE-cadherin expression (Fig. 3d, panel iii).
To identify the contribution of PMA and S1P to angiogenic sprouting, we directly compared VEGF + PMA with VEGF + S1P. The combination of VEGF + PMA triggered the formation of angiogenic sprouts into the gel, but the tip cells fail to develop their characteristic tip cell morphology including filopodia and the sprouts lack directionality after 6 days of sprouting (Fig. 3e and Supplementary Fig. 3a, b). Furthermore, the sprouts appear to be non-homogenously distributed within the collagen gel. In contrast, VEGF + S1P shows sprouts that are also connected the sprouts to the main vessel, but sprouts are equally distributed within the gel with a clear directionality towards the gradient (Fig. 3f). Although there were not significantly more sprouts after VEGF + S1P stimulation (Fig. 3g), the diameter of the sprouts was significantly lower (Fig. 3h). We quantified the circularity of the sprouts to estimate the directionality: a perpendicular sprout appears circular in a cross-sectional view with a value closer to 1, while a deviating sprout appears flattened (closer to 0). This shows that VEGF + S1P sprouts have a significantly higher circularity and thus improved directionality towards the gradient compared to VEGF + PMA (Fig. 3i). Taken together, these results clearly demonstrate that in a gradient-driven, 3D cell culture environment, a combination of different cues is required to trigger angiogenesis, and S1P is a crucial factor in the distribution and guidance during angiogenic sprouting.
Anastomosis triggers remodeling and stabilization
Prolonged exposure to growth factors caused the angiogenic sprouts to anastomose, and connection is formed between the two perfusion channels. After anastomosis, we observed a significant reduction of sprouts (Fig. 4a, b). Some angiogenic sprouts display the characteristic steps involved in pruning: first, the lumen collapses, followed by regression of the angiogenic sprouts towards the parental vessel (Fig. 4a, b, arrows), while other angiogenic sprouts remained and increased their lumen diameter (Fig. 4a, b arrowheads).
The formation of perfusable lumen within the sprouts is visualized by perfusion of the main vessel with 0.5 mg/mL 150 kDa TRITC-Dextran (Fig. 4c, d). A surplus of 50 µL is added to the inlet well, which fills the parental vessels and flows into the angiogenic sprouts. When angiogenic sprouts did not connect to the basal perfusion channel (Fig. 4c), spots were visible within the collagen where dextran leaks out of the tip of the sprouts (panel ii, left, 0 min). These spots increased over time (right, 9 min). However, after anastomosis (Fig. 4d), sprouts retained the dextran in their lumen, and shows subsequent filling of the bottom basal perfusion channel. This shows that sprouts stabilize and form a functional barrier after a connection has been formed.