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

Perfused 3D angiogenic sprouting in a high-throughput in vitro platform 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 cur- rent 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 endothe- lial 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 Microfluidics · Angiogenic sprouting · Vascular stabilization · In vitro · 3D cell culture Introduction The loss of vascular integrity plays a rate-limiting role in the onset and progression of diseases such as arteriosclero- sis and cancer and conditions such as chronic inflammation and ischemia [1, 2]. Therefore, detailed knowledge of the Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1045 6-018-9647-0) contains mechanisms of microvascular loss or the formation of novel supplementary material, which is available to authorized users. vascular structures such as those generated by angiogenesis are of major importance. * V. van Duinen Endothelial cells (ECs) respond to pro-angiogenic stimuli [email protected] by differentiating into three characteristic phenotypes: tip, Division of Analytical Biosciences, LACDR, Leiden stalk, and phalanx cells [3–6]. Each of these phenotypes has University, Leiden, The Netherlands a specific function in the development and maturation of the Mimetas BV, Leiden, The Netherlands newly formed vasculature, and its differentiation from ECs The Department of Internal Medicine, division is tightly coordinated and regulated in order to achieve func- of Nephrology and the Einthoven Laboratory for Vascular tional, luminized vascular networks. After formation of a and Regenerative Medicine, LUMC, Leiden, The Netherlands Vol.:(0123456789) 1 3 158 Angiogenesis (2019) 22:157–165 pre-mature vascular network, perfusion of the newly formed as flow [16] and spatial–temporal gradients [17]. Micro- capillary initiates the final phase of angiogenesis: stabili- fluidics is an important emerging technique to facilitate zation of the vascular network through an increase in the 3D-cell culture models aimed to more faithfully mimic adherence junctions, pruning of the non-functional sprouts, tissue architecture [18]. For instance, a number of micro- and pericyte attraction to the vascular network [7–11]. fluidic devices for microvascular modeling have been pre- In vitro models are essential to study angiogenesis in a sented that allow lumen perfusion [19–30]. For an increas- defined and well-controlled environment. Two-dimensional ing number of research laboratories that study angiogenesis in vitro models allow the study of fundamental EC biol- such microfluidic platforms are becoming their method of ogy in high-throughput, such as migration and proliferation choice (Table 1) [31]. However, most microfluidic assays are [12]. However, since these models lack a more physiologic, limited in terms of scalability, standardization, and usability three-dimensional environment, the endothelial cells fail to [32]. Many microfluidic devices need to be manufactured show many of the typical hallmarks of endothelial cells dur- manually before use, which strongly limits routine adoption ing angiogenesis in vivo [13], such as lumen formation and [33]. Furthermore, many prototypes show limited through- differentiation into tip and stalk cells. 3D cell culture mod- put per assay (n < 8) [18, 20, 34] and require tubings and els with EC growing within a matrix such as fibrin display pumps which increases complexity and limits scalability of a higher level of physiological relevance, as ECs are able these platforms. to degrade the extracellular matrix, form lumen and show Here, we report a standardized, high-throughput cell cul- anastomosis between adjacent sprouts [14, 15]. Nonethe- ture platform to study angiogenesis. The platform consists less, as such 3D cell culture models have EC mixed with of an array of 40 microfluidic devices, integrated underneath an extracellular matrix, the formed lumen is not accessible a 384-well plate. This format is compatible with standard or perfusable. Furthermore, possibilities to apply a stable (high content) imaging equipment. It enables the culture of gradient of growth factors to direct the formation of capil- individually addressable, perfusable microvessels against a laries are limited. patterned, three-dimensional matrix or hydrogel. To elimi- Microfluidic devices have micrometer-sized channels nate the need for pumps while increasing the robustness that enable spatial control over cells and matrices and allow and scalability, passive leveling is used as a source of flow. the incorporation of important biological parameters such Within this platform, reproducible gradients can be formed Table 1 Comparison of in vitro assays to study angiogenesis Type Assay Strengths Weaknesses References 2D Scratch Easy to perform Lacks soft substrate for the cells [12] Easy to quantify Migration is in 2D Tube formation Cells adhere to soft substrate No distinct tip/stalk cell phenotype [13] Self-organization into cords Basement membrane extracts contain significant Reasonable throughput levels of growth factors and have a high batch- Tools are available for quantification to-batch variability Limited tube survival (< 2 days) High use of reagents compared to microfluidic assays Lumens not accessible nor perfusable 3D Spheroid Cells grow in 3D in a soft supportive matrix Lacks spatial control over gradients [14, 15, 40] Endothelial cells differentiate into tip and stalk Higher use of reagents compared to microfluidic cells assays Clear lumen formation Spheroids are randomly distributed throughout Fusion of sprouts is observed gel/matrix Laser dissection allows capture of cells Lumens are not accessible nor perfusable Tools available to quantify the angiogenic sprouts Microfluidic Biochemical gradients can be created and main- Some devices require for pumps to supply flow [18–30, 32, 33] tained and maintain gradients Lumen formation occurs early (more comparable  Handling and scalability issues due incompatibil- to in vivo) ity with other equipment Angiogenic sprouts can be perfused Some devices need to be manufactured by the Spatial control over multiple cells (e.g., fibro- end-user blasts, pericytes) Biocompatibility of the used materials Lack of standardization Limited possibilities to extract a subset of cells 1 3 Angiogenesis (2019) 22:157–165 159 and maintained for multiple days. Since gradients and perfu- Stimulation with angiogenic factors sion are two important cues during the initial sprouting and the stabilization phase in angiogenesis [3, 35], the integra- Microvessels were first cultured for 3 days before any gra - tion of these cues in our novel platform technology makes dients of growth factors were applied. Growth factors were our model uniquely suited to perform physiologically rel- replaced every 2–3 days. Stock solutions were prepared as evant studies on the formation and regression of the micro- following: 50 µg/mL murine VEGF in MilliQ water (Pre- vasculature in vitro. protech, 450-32), 20 ng/mL bFGF in MilliQ water (Pepro- tech, 100-18B), 1 mM Sphingosine-1-Phosphate (Sigma, S9666) in 5% 1 M HCl, 95% DMSO, and 2 µg/mL PMA Methods (Sigma, P1585) in 1% DMSO. Angiogenic factors were diluted in MV2 culture medium and used in the following Cell culture concentrations: 50 ng/mL for VEGF, 50 ng/mL for bFGF, 2 ng/mL for PMA, and 500 nM for S1P. HUVEC-VeraVec™ human endothelial cells (Angiocrine Biosciences, hVera101) were cultured in T75 flasks (Nunc™ EasyFlask, Sigma F7552) with endothelial Cell Growth Sprout permeability visualization Medium MV2 (Promocell, C-22022) and used at P3 till P9. Media was replaced three times a week. Cells tested negative Angiogenic sprouts were stimulated with for mycoplasma. All cell culture was performed in a humidi- VEGF + bFGF + PMA + S1P for 9 days. At day 4 and day fied incubator at 37 °C and 5% CO . 9 after stimulation, 50 µL of a 150 kDa TRITC-Dextran (Sigma 48946) solution (0.5  mg/mL in MV2 culture Microfluidic cell culture media) was added to the perfusion inlet well and time- lapse images were acquired at 1 min intervals using the 3-Lane microfluidic titer plates (MIMETAS OrganoPlates × 10 objective. 4003-400B) were used for all microfluidic cell culture. Before gel seeding, every center well was filled with 50 µL hanks balanced salt solution (HBSS) to provide optical clar- Immunocytofluorescent staining ity and prevention of gel dehydration. Collagen type I (R&D systems, 3447-020-01) was used as 3D scaffold. A stock During all steps of the immunofluorescent staining, the solution of 5 mg/mL rat tail collagen type I was neutralized device is placed under an angle to create flow, except dur - with 10% 37 g/L N aHCO (Sigma, S5761) and 10% 1 M ing staining with primary antibody. All solutions were HEPES buffer (Gibco, 15630-056) to obtain a concentra- used in quantities of 50 µL per every inlet and outlet well, tion of 4 mg/mL. The neutralized collagen was kept on ice unless specified otherwise. Cells were fixed using freshly until use and used within 30 min. Using a repeater pipette, prepared 3.7% formaldehyde (Sigma 252549) in PBS. 2 µL of the neutralized collagen was added into the inlet of 50 µL of the fixative was added to both the perfusion inlet each gel channel. To polymerize the collagen, the device was and outlet for 15 min at room temperature (RT), followed incubated for 10 min at 37 °C, 5% CO . After incubation, the by a wash step with 4% FBS in PBS for 5 min. After fixa- device was removed from the incubator and kept sterile at tion, the cells were permeabilized using 0.3% Triton-X room temperature right before cell loading. Endothelial cells (Sigma T8787) in PBS. After washing, the microvessels were dissociated, pelleted, and suspended in MV2 medium were blocked for 45 min using blocking solution (2% FBS, in a concentration of 2 × 10  cells/mL. 2 µL of the cell sus- 0.1% Tween20 (Sigma P9169), 2% BSA (Sigma A2153) pension was dispensed into the perfusion inlet and incubated in PBS). The adherence junctions were visualized using for 45 min at 37 °C, 5% C O . After the cells attached to a VE-Cadherin stain (Abcam, 33168, diluted 1:1000 in the bottom of the perfusion channel, 50 µL of medium was blocking solution, 30 µL pipetted in the perfusion inlet, added in the perfusion inlet and outlet wells and the plates 20 µL in the perfusion outlet), which was incubated for were placed on an interval rocker platform for continuous 1 h at RT followed by 30-min incubation with Alexa Fluor perfusion. (Perfusion rocker, MIMETAS). The rocker was 488 (ThermoFisher Scientific, A11008, 1:250 in block - set at a 7-degree inclination and 8-min cycle time. Medium ing solution). To perfuse the chips with primary antibody, was refreshed three times a week. the device was placed on a rocker platform. After incuba- tion with the secondary antibody, the device is washed once with washing solution, followed by nuclei staining (NucBlue Fixed cell staining, Life technologies, R37606), and the cytoskeletal marker F-actin, stained by ActinRed™ 1 3 160 Angiogenesis (2019) 22:157–165 555 ReadyProbes® (ThermoFisher Scientific, R37112) in Results PBS and imaged using a high content confocal microscope (Molecular Devices, ImageXpress™ Micro Confocal) at Robust gradient formation in a 3D 10x magnification. microenvironment Sprouting quantification The microfluidic culture platform is based on a 384-well microtiter plate format. The glass bottom contains 40 micro- The average sprouting length was quantified using FIJI v. fluidic units (Fig.  1a), and each microfluidic unit is posi - 1.52 by manual determination of the distance between the tioned underneath nine wells (3 × 3). Every unit consists of microvessel and the tip cell sprouting furthest into the gel. three channels: the center channel that is used to pattern The sprouting length of PMA was obtained after 3 days, an  extracellular matrix (‘gel channel’) and two adjacent all other combinations after 4  days. VEGF + PMA and channels (‘perfusion channels’) (Fig. 1b). The channels are VEGF + S1P microvessels after 6 days of stimulation were separated by PhaseGuides: small ridges that function as cap- used to quantify the median sprout number, average diam- illary pressure barriers, which enable patterning of cells and eter in the minor direction, and circularity. Images were gel without the use of artificial membranes [37]. Every chan- obtained from two replicates for every condition. Using nel has one inlet and one outlet, which connect the channels a 10x objective, we acquired 180 z-steps with 1 µm spac- with the wells in the microtiter plate. Compartmentalization ing and obtained two adjacent sites. The orthogonal views is achieved by patterning a hydrogel in the middle channel were extracted and analyzed in the middle of the gel region. (Fig. 1c, step 1), and enables the formation of gradients by Thresholding of the vessels was automated using Weka Seg- adding a source and sink in the opposite perfusion channels mentation tool [36] (v 3.2.27). Particle analysis was per- (Fig. 1c, step 2). Without continuous replenishment of the formed to include particles between 10 and 10,000 µm with gradients source and sink in the microfluidic channels, gra- a circularity between 0.10 and 1.00. dients 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 continu- ously and simultaneously (Fig. 1c, step 3). As the volume inside the wells is typically orders of a magnitude higher Fig. 1 Gradient generation in a 3D microenvironment. a Bottom of Three-step method to generate gradients in patterned hydrogels. Step the OrganoPlate®, a microfluidic culture platform based on a 384- 1: 2 µL of collagen-1 gel is added in the center channel and polymer- well plate. The glass bottom includes 40 microfluidic devices. b The ized. Step 2: source and sink are added in opposite perfusion chan- geometry of a single microfluidic device that is positioned underneath nels. Step 3: the device is placed on a rocker platform to perfuse both nine wells (3 × 3). Every device consists of three channels: one ‘gel’ perfusion channels continuously to generate a gradient. d Gradient channel for gel patterning, and two adjacent channels. Phaseguides visualization after 1, 3, and 6  days after addition of 20  kDa FITC- prevent the patterned gel from flowing into the adjacent channels. c Dextran as a gradient source 1 3 Angiogenesis (2019) 22:157–165 161 than in the microfluidic channels (the wells typically contain the perfusion channel, while the gel forms the basal side of volumes of 50 µL, compared to < 1 µL in the microfluidic the tube [38]. channels), the source and sink within the microfluidic chan- nels are constant over prolonged periods of time. Thus, a sta- Combination of angiogenic factors is required ble gradient could be maintained for multiple days (Fig. 1d) to induce sprouting without the need to replenish. Although a gradient is still present after 6 days, the steepness is affected due to satura- After reaching confluency in 3  days, the microvessels tion of the sink. Therefore, growth factors and medium were showed a stable morphology of a single monolayer against replaced at 2–3-day time intervals. the gel (Fig. 2b, step 1), despite the numerous (angiogenic) Importantly, the high hydraulic resistance of the hydrogel growth factors that are present in the media (such as vas- limits the influence of differences in hydrostatic pressures. cular endothelial growth factor (VEGF) and basic fibro- This results in a reproducible and robust platform to gener- blast growth factor (bFGF)). We included VEGF and S1P ate gradients, despite the presence of small difference in as they have been shown to induce angiogenic sprouting volumes, for example, due to pipetting errors. Nonetheless, within a collagen-1 matrix [39–41] and included phorbol hydrostatic pressures still can influence the shape of the 12-myristate 13-acetate (PMA) as it has been found to pro- gradient, when the difference between the volumes is suf- mote lumen formation in the absence of fibroblasts [15, 42], ficiently large. This allows different types of gradient to be and used in concentrations of 50 ng/mL for VEGF, 500 nM generated (e.g., linear or parabolic, Supplementary Fig. 1). for S1P, and 2 ng/mL for PMA. The angiogenic growth fac- tor cocktail was added on the basal side of the vessels, and Microvessels cultured against patterned collagen‑1 formed a gradient within the collagen-1 gel (Fig. 2b, step gel 1). This induced the formation of tip and stalk cells after respectively 1 and 2 days (Fig. 2b, step 2–3). After gel loading and polymerization (Fig.  2a, step 1), Interestingly, adding either VEGF, S1P, or PMA alone endothelial cell suspensions were added to the perfusion on the basal side did not result in angiogenic sprouting channels adjacent to the gel. After the cells adhered to the (Supplementary Fig.  2). We quantified the angiogenesis glass substrate (step 2) of the channel, perfusion was applied after addition of various combinations of VEGF, PMA and by placing the device on a rocker platform (step 3). Conflu- S1P (Fig. 3a, b). VEGF + PMA + S1P together  resulted in ent microvessels were formed after 3 days of culture, and the angiogenesis including tip/stalk cell formation, the presence apical side of the vessel (the lumen) can be accessed through of filopodia and lumen formation and directional growth towards the gradient. The sprouts fully traversed the gel after Fig. 2 Microvessel culture against a  patterned collagen-1 gel. a was formed. b Angiogenesis assay using a gradient of angiogenic fac- Method the culture a microvessel within a microfluidic device. First, tors. Angiogenic factors are added once a stable monolayer of ECs is collagen-1 gel is patterned in the middle channel. After polymeriza- formed against the gel (step 1). Addition of a gradient of angiogenic tion, an endothelial cell suspension was added in the adjacent perfu- growth factors resulted in tip cells formation including filopodia at sion channel. By placing the device on a rocker platform, the chan- day 1 (step 2). Lumens formed by the stalk cells are visible at day 2 nels are continuously perfused. After 72  h, a confluent microvessel (step 3) 1 3 162 Angiogenesis (2019) 22:157–165 Fig. 3 Angiogenic sprouts after addition of angiogenic factors. a Stained against F-actin (red) and nucleus (blue) and VE-cadherin Images of sprouting after 4  days of stimulation of a gradient of dif- (green). e Same as c, but stimulation with VEGF + PMA. f Same ferent combinations of angiogenic factors. b Quantification of maxi- as c, but stimulation with VEGF + S1P. g–i Comparison between mum absolute sprouting length in µm after stimulation for 3 (PMA) VEGF + PMA and VEGF + S1P in number of sprouts, diameter, or 4  days (all other combinations) (n = 6). c Angiogenic sprouts and circularity (n = 2). Significance was calculated using one-way after 6 days of stimulation with VEGF + PMA + S1P, stained against anova (b) or Student’s t test (g–i) and shown as n.s (non-significant), F-actin (red) and nucleus (blue). d Close-up of middle (i), top (ii), *(P < 0.05), **(P < 0.01), or ***(P < 0.001). Scale bars: 100  µm. and cross-section (iii) of VEGF + PMA + S1P stimulated sprouting. Graphs are presented as mean ± SD about 6 days and started to form a continuous monolayer towards the gradient compared to VEGF + PMA (Fig. 3i). against in the channel on the other side of the gel and in the Taken together, these results clearly demonstrate that in a basal perfusion channel  (Fig. 3c). The angiogenic sprouts gradient-driven, 3D cell culture environment, a combination have a clear lumen formation (Fig. 3d, panel i), appear cir- of different cues is required to trigger angiogenesis, and S1P cular in a cross-sectional view (Fig. 3d, panel ii), and have is a crucial factor in the distribution and guidance during clear VE-cadherin expression (Fig. 3d, panel iii). angiogenic sprouting. To identify the contribution of PMA and S1P to angio- genic sprouting, we directly compared VEGF + PMA with Anastomosis triggers remodeling and stabilization VEGF + S1P. The combination of VEGF + PMA triggered the formation of angiogenic sprouts into the gel, but the tip Prolonged exposure to growth factors caused the angiogenic cells fail to develop their characteristic tip cell morphology sprouts to anastomose, and connection is formed between the including filopodia and the sprouts lack directionality after two perfusion channels. After anastomosis, we observed a 6 days of sprouting (Fig. 3e and Supplementary Fig. 3a, b). significant reduction of sprouts (Fig.  4a, b). Some angiogenic Furthermore, the sprouts appear to be non-homogenously sprouts display the characteristic steps involved in pruning: distributed within the collagen gel. In contrast, VEGF + S1P first, the lumen collapses, followed by regression of the angio- shows sprouts that are also connected the sprouts to the main genic sprouts towards the parental vessel (Fig. 4a, b, arrows), vessel, but sprouts are equally distributed within the gel with while other angiogenic sprouts remained and increased their a clear directionality towards the gradient (Fig. 3f). Although lumen diameter (Fig. 4a, b arrowheads). there were not significantly more sprouts after VEGF + S1P The formation of perfusable lumen within the sprouts is stimulation (Fig. 3g), the diameter of the sprouts was sig- visualized by perfusion of the main vessel with 0.5 mg/mL nificantly lower (Fig.  3h). We quantified the circularity of 150 kDa TRITC-Dextran (Fig. 4c, d). A surplus of 50 µL is the sprouts to estimate the directionality: a perpendicular added to the inlet well, which fills the parental vessels and sprout appears circular in a cross-sectional view with a value flows into the angiogenic sprouts. When angiogenic sprouts closer to 1, while a deviating sprout appears flattened (closer did not connect to the basal perfusion channel (Fig. 4c), spots to 0). This shows that VEGF + S1P sprouts have a signifi - were visible within the collagen where dextran leaks out of the cantly higher circularity and thus improved directionality tip of the sprouts (panel ii, left, 0 min). These spots increased 1 3 Angiogenesis (2019) 22:157–165 163 Fig. 4 Anastomosis with basal channel triggers pruning and matura- obtained every minute and directly after addition of a 0.5  mg/mL tion of angiogenic sprouts. a Angiogenic sprouts 5  days after addi- 150 kDa TRITC-Dextran solution in culture media. Panel ii shows the tion of VEGF + PMA + S1P. Compared to the angiogenic sprouts at pseudo-colored fluorescent images after 0 and 9 min after addition of day 8. b Some sprouts regressed (arrows) while other sprouts remain the dextran solutions. Time is indicated in min. d Same as in c, but and showed increased lumen diameter (arrowheads). c Angiogenic after 9  days of stimulation. Sprouts are connected to the other side sprouts after 4 days of stimulation invaded into the gel but are not yet and formed a confluent microvessel in the basal perfusion channel. connected to the bottom perfusion channel. Fluorescent images were Scale bars: 100 µm over time (right, 9 min). However, after anastomosis (Fig. 4d), important mechano-biological signal in during angiogen- sprouts retained the dextran in their lumen, and shows subse- esis [43], while flow in this assay occurs at discrete time quent l fi ling of the bottom basal perfusion channel. This shows points and is bi-directional. Thus, despite the evidence that that sprouts stabilize and form a functional barrier after a con- flow affects the remodeling and maturation of the capil - nection has been formed. laries in our model, the exact contribution of flow in this assay is difficult to determine. We showed that gradient-driven angiogenic sprouting Discussion through an extracellular matrix requires not just the pres- ence of VEGF, but a combination of multiple angiogenic We report a robust, standardized microfluidic cell cul- factors [44]. The combination of VEGF + PMA + S1P was ture platform to study gradient-driven angiogenesis of the most optimal cocktail to trigger quick, robust, directional a perfused microvessel in high-throughput. Each device angiogenesis with angiogenic sprouts with clear lumen for- contains 40 individually addressable microfluidic units mation. VEGF + PMA showed a random distribution of the and enables the culture of 40 identical microvessels. An sprouts and an absence of filopodia on the tips cells, and important advantage of this assay is the defined geometry the sprouts lacked directionality. In contrast, a VEGF + S1P of the microfluidic channels, as this results in reproducible gradient showed formation of angiogenic sprouts, includ- experimental cell culture conditions (position and density ing tip cells with filopodia. Filopodia allow the tip cells to of the cells, amount of flow, position of the extracellular sense a biochemical gradient [4], and explains the observed matrix and the shape of the gradient) and increases the directionality of the angiogenic sprouts. This suggests that robustness and scalability of our assay. S1P plays an important role in the differentiation into func- Perfusion in our device is induced by passive leveling tional tip cells and the observed repetitive formation of angi- using a rocker platform, and has two crucial advantages. ogenic sprouts. Such a repetitive formation of angiogenic First, the flow is simultaneously applied throughout all sprouts can be explained by a reaction–diffusion mecha- microfluidic units, which results in reproducible gradient nism between VEGF and Flt-1, the soluble form of VEGF formation. Second, as tubing and pumps are not required receptor. Stalk cells are known to secrete Flt-1, which binds the throughput is greatly increased: the assay is scalable VEGF and prevents neighboring cells to become tip cells since multiple experiments can be performed by stacking [45]. This is required for efficient angiogenic sprouting into of culture platforms on top of each other. Nonetheless, the matrix [3], with evenly distributed sprouts roughly every using a rocker platform to induce flow is also a trade- 100 µm, as predicted in silico [8, 9]. It has been shown that off that has its downsides: first, the requirement of a S1P has a pro-angiogenic effect in vitro [39, 40, 46–48] and rocker platform limits us to perform time-lapse imaging in vivo [39, 49, 50]. Our data suggest a pro-angiogenic syn- only at discrete time points, as the vessels and gradient ergy between S1P and VEGF, which is in agreement with require continuous perfusion. Second, vasculature in vivo the fact that inhibition of S1P also prevents VEGF-induced is exposed to continuous, unidirectional flow that is an angiogenesis in vivo [51]. Interestingly, S1P is also known 1 3 164 Angiogenesis (2019) 22:157–165 for its barrier stabilizing, anti-angiogenic properties, and Conclusion vascular maturation [52, 53]. Therefore, we hypothesize that the effect of S1P is dependent on whether it is present We demonstrate a gradient-driven, three-dimensional angio- on the apical side of ECs (lumen) or basal side, either medi- genesis assay in a standardized microfluidic platform. Angi- ated by differences in apical and basal expression of S1P ogenic sprouting is induced from a perfused microvessel receptors [54] or by dimerization with other receptors, like through a patterned collagen-1 gel. The combination of angi- basally expressed VEGFR2 [46]. A better understanding of ogenic factors was optimized to trigger angiogenic sprout- the precise mechanisms of S1P signaling in angiogenesis ing that faithfully reproduces all the angiogenic events that will provide therapeutic strategies that specifically target the occur in vivo, such as the differentiation of the endothelial pro-angiogenic effects of S1P [49]. cells into tip, stalk, and phalanx cells and the formation of Prolonged exposure (> 6 days) to a gradient of angio- perfusable lumen. It was found that a combination of VEGF, genic stimuli resulted in sprouts that connect the two perfu- S1P, and PMA provided the optimal cocktail for 3D angio- sion channels (anastomosis). This connection resolves the genic sprouting. After the angiogenic sprouts anastomosed gradient, as there is a direct connection between the source through the collagen to the other channel, remodeling and and sink, and also results in the onset of flow through the stabilization of the capillary bed was observed. sprouts. There remains controversy about the exact mecha- Acknowledgements We thank Angiocrine for the kind gift of VeraVec nism that leads to pruning. In vivo, this is either shear-medi- HUVEC cells. V. van Duinen was partially financially supported by ated or due to changing receptor expression after a resolved the STW Valorisation Grant (STW 12615), the VIRGO consortium (oxygen) gradient [10, 55]. Once anastomosis occurred, we (FES0908), and supported bythe Dutch Heart Foundation (CVON observed remodeling of the capillary bed, including pruning RECONNECT) and ZonMw (MKMD:114022501) Grant to T. Hanke- meier and A. J. van Zonneveld. and regression of angiogenic sprouts within the collagen. Furthermore, some sprouts increased in lumen diameter, Author contributions VD, CR and DZ performed the experiments. VD likely caused by the onset of perfusion [56]. By controlling wrote the manuscript with input from all authors. PV, TH and AJZ shear levels and oxygen tension in this assay, we will be able supervised all aspects of the work. to determine which of those effects is the crucial mechanism in pruning. Compliance with ethical standards Perfusion of the sprouts with fluorescently labeled dex- tran showed that angiogenic sprouts that did anastomose are Conflict of interest P. Vulto and T. Hankemeier are shareholders in Mimetas BV. V. van Duinen, D. Zhu, C. Ramakers and A. J. van Zonn- permeable near the tip/stalk cell region. In contrast, anasto- eveld declare no potential conflict of interest. mosed sprouts retained the 150 kDa dextran solution within their lumen, suggesting that the connection between the two Open Access This article is distributed under the terms of the Crea- channels triggers maturation of the ECs in the sprouts, as tive Commons Attribution 4.0 International License (http://creat iveco they adopt their characteristic phalanx phenotype including mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate mature cell–cell junctions [55, 57]. Furthermore, once the credit to the original author(s) and the source, provide a link to the angiogenic sprouts connected, the medium can be switched Creative Commons license, and indicate if changes were made. back to the original culture medium with low levels or growth factors, while the integrity of the sprouts remained (Supplementary movies 2, 3), which suggests that perfusion is an important survival factor for angiogenic sprouts in the References absence of a high concentration of angiogenic factors like VEGF. 1. Carmeliet P (2005) Angiogenesis in life, disease and medicine. We expect that our platform will be widely adopted for a Nature 438(7070):932–936 2. Carmeliet P, Jain RK (2000) Angiogenesis in cancer and other range of applications, including both fundamental studies of diseases. Nature 407(6801):249–257 the mechanisms of angiogenesis as well as for the identic fi a - 3. 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Perfused 3D angiogenic sprouting in a high-throughput in vitro platform

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Springer Journals
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Copyright © 2018 by The Author(s)
Subject
Biomedicine; Cancer Research; Biomedicine, general; Cell Biology; Cardiology; Ophthalmology; Oncology
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0969-6970
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1573-7209
DOI
10.1007/s10456-018-9647-0
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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 cur- rent 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 endothe- lial 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 Microfluidics · Angiogenic sprouting · Vascular stabilization · In vitro · 3D cell culture Introduction The loss of vascular integrity plays a rate-limiting role in the onset and progression of diseases such as arteriosclero- sis and cancer and conditions such as chronic inflammation and ischemia [1, 2]. Therefore, detailed knowledge of the Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1045 6-018-9647-0) contains mechanisms of microvascular loss or the formation of novel supplementary material, which is available to authorized users. vascular structures such as those generated by angiogenesis are of major importance. * V. van Duinen Endothelial cells (ECs) respond to pro-angiogenic stimuli [email protected] by differentiating into three characteristic phenotypes: tip, Division of Analytical Biosciences, LACDR, Leiden stalk, and phalanx cells [3–6]. Each of these phenotypes has University, Leiden, The Netherlands a specific function in the development and maturation of the Mimetas BV, Leiden, The Netherlands newly formed vasculature, and its differentiation from ECs The Department of Internal Medicine, division is tightly coordinated and regulated in order to achieve func- of Nephrology and the Einthoven Laboratory for Vascular tional, luminized vascular networks. After formation of a and Regenerative Medicine, LUMC, Leiden, The Netherlands Vol.:(0123456789) 1 3 158 Angiogenesis (2019) 22:157–165 pre-mature vascular network, perfusion of the newly formed as flow [16] and spatial–temporal gradients [17]. Micro- capillary initiates the final phase of angiogenesis: stabili- fluidics is an important emerging technique to facilitate zation of the vascular network through an increase in the 3D-cell culture models aimed to more faithfully mimic adherence junctions, pruning of the non-functional sprouts, tissue architecture [18]. For instance, a number of micro- and pericyte attraction to the vascular network [7–11]. fluidic devices for microvascular modeling have been pre- In vitro models are essential to study angiogenesis in a sented that allow lumen perfusion [19–30]. For an increas- defined and well-controlled environment. Two-dimensional ing number of research laboratories that study angiogenesis in vitro models allow the study of fundamental EC biol- such microfluidic platforms are becoming their method of ogy in high-throughput, such as migration and proliferation choice (Table 1) [31]. However, most microfluidic assays are [12]. However, since these models lack a more physiologic, limited in terms of scalability, standardization, and usability three-dimensional environment, the endothelial cells fail to [32]. Many microfluidic devices need to be manufactured show many of the typical hallmarks of endothelial cells dur- manually before use, which strongly limits routine adoption ing angiogenesis in vivo [13], such as lumen formation and [33]. Furthermore, many prototypes show limited through- differentiation into tip and stalk cells. 3D cell culture mod- put per assay (n < 8) [18, 20, 34] and require tubings and els with EC growing within a matrix such as fibrin display pumps which increases complexity and limits scalability of a higher level of physiological relevance, as ECs are able these platforms. to degrade the extracellular matrix, form lumen and show Here, we report a standardized, high-throughput cell cul- anastomosis between adjacent sprouts [14, 15]. Nonethe- ture platform to study angiogenesis. The platform consists less, as such 3D cell culture models have EC mixed with of an array of 40 microfluidic devices, integrated underneath an extracellular matrix, the formed lumen is not accessible a 384-well plate. This format is compatible with standard or perfusable. Furthermore, possibilities to apply a stable (high content) imaging equipment. It enables the culture of gradient of growth factors to direct the formation of capil- individually addressable, perfusable microvessels against a laries are limited. patterned, three-dimensional matrix or hydrogel. To elimi- Microfluidic devices have micrometer-sized channels nate the need for pumps while increasing the robustness that enable spatial control over cells and matrices and allow and scalability, passive leveling is used as a source of flow. the incorporation of important biological parameters such Within this platform, reproducible gradients can be formed Table 1 Comparison of in vitro assays to study angiogenesis Type Assay Strengths Weaknesses References 2D Scratch Easy to perform Lacks soft substrate for the cells [12] Easy to quantify Migration is in 2D Tube formation Cells adhere to soft substrate No distinct tip/stalk cell phenotype [13] Self-organization into cords Basement membrane extracts contain significant Reasonable throughput levels of growth factors and have a high batch- Tools are available for quantification to-batch variability Limited tube survival (< 2 days) High use of reagents compared to microfluidic assays Lumens not accessible nor perfusable 3D Spheroid Cells grow in 3D in a soft supportive matrix Lacks spatial control over gradients [14, 15, 40] Endothelial cells differentiate into tip and stalk Higher use of reagents compared to microfluidic cells assays Clear lumen formation Spheroids are randomly distributed throughout Fusion of sprouts is observed gel/matrix Laser dissection allows capture of cells Lumens are not accessible nor perfusable Tools available to quantify the angiogenic sprouts Microfluidic Biochemical gradients can be created and main- Some devices require for pumps to supply flow [18–30, 32, 33] tained and maintain gradients Lumen formation occurs early (more comparable  Handling and scalability issues due incompatibil- to in vivo) ity with other equipment Angiogenic sprouts can be perfused Some devices need to be manufactured by the Spatial control over multiple cells (e.g., fibro- end-user blasts, pericytes) Biocompatibility of the used materials Lack of standardization Limited possibilities to extract a subset of cells 1 3 Angiogenesis (2019) 22:157–165 159 and maintained for multiple days. Since gradients and perfu- Stimulation with angiogenic factors sion are two important cues during the initial sprouting and the stabilization phase in angiogenesis [3, 35], the integra- Microvessels were first cultured for 3 days before any gra - tion of these cues in our novel platform technology makes dients of growth factors were applied. Growth factors were our model uniquely suited to perform physiologically rel- replaced every 2–3 days. Stock solutions were prepared as evant studies on the formation and regression of the micro- following: 50 µg/mL murine VEGF in MilliQ water (Pre- vasculature in vitro. protech, 450-32), 20 ng/mL bFGF in MilliQ water (Pepro- tech, 100-18B), 1 mM Sphingosine-1-Phosphate (Sigma, S9666) in 5% 1 M HCl, 95% DMSO, and 2 µg/mL PMA Methods (Sigma, P1585) in 1% DMSO. Angiogenic factors were diluted in MV2 culture medium and used in the following Cell culture concentrations: 50 ng/mL for VEGF, 50 ng/mL for bFGF, 2 ng/mL for PMA, and 500 nM for S1P. HUVEC-VeraVec™ human endothelial cells (Angiocrine Biosciences, hVera101) were cultured in T75 flasks (Nunc™ EasyFlask, Sigma F7552) with endothelial Cell Growth Sprout permeability visualization Medium MV2 (Promocell, C-22022) and used at P3 till P9. Media was replaced three times a week. Cells tested negative Angiogenic sprouts were stimulated with for mycoplasma. All cell culture was performed in a humidi- VEGF + bFGF + PMA + S1P for 9 days. At day 4 and day fied incubator at 37 °C and 5% CO . 9 after stimulation, 50 µL of a 150 kDa TRITC-Dextran (Sigma 48946) solution (0.5  mg/mL in MV2 culture Microfluidic cell culture media) was added to the perfusion inlet well and time- lapse images were acquired at 1 min intervals using the 3-Lane microfluidic titer plates (MIMETAS OrganoPlates × 10 objective. 4003-400B) were used for all microfluidic cell culture. Before gel seeding, every center well was filled with 50 µL hanks balanced salt solution (HBSS) to provide optical clar- Immunocytofluorescent staining ity and prevention of gel dehydration. Collagen type I (R&D systems, 3447-020-01) was used as 3D scaffold. A stock During all steps of the immunofluorescent staining, the solution of 5 mg/mL rat tail collagen type I was neutralized device is placed under an angle to create flow, except dur - with 10% 37 g/L N aHCO (Sigma, S5761) and 10% 1 M ing staining with primary antibody. All solutions were HEPES buffer (Gibco, 15630-056) to obtain a concentra- used in quantities of 50 µL per every inlet and outlet well, tion of 4 mg/mL. The neutralized collagen was kept on ice unless specified otherwise. Cells were fixed using freshly until use and used within 30 min. Using a repeater pipette, prepared 3.7% formaldehyde (Sigma 252549) in PBS. 2 µL of the neutralized collagen was added into the inlet of 50 µL of the fixative was added to both the perfusion inlet each gel channel. To polymerize the collagen, the device was and outlet for 15 min at room temperature (RT), followed incubated for 10 min at 37 °C, 5% CO . After incubation, the by a wash step with 4% FBS in PBS for 5 min. After fixa- device was removed from the incubator and kept sterile at tion, the cells were permeabilized using 0.3% Triton-X room temperature right before cell loading. Endothelial cells (Sigma T8787) in PBS. After washing, the microvessels were dissociated, pelleted, and suspended in MV2 medium were blocked for 45 min using blocking solution (2% FBS, in a concentration of 2 × 10  cells/mL. 2 µL of the cell sus- 0.1% Tween20 (Sigma P9169), 2% BSA (Sigma A2153) pension was dispensed into the perfusion inlet and incubated in PBS). The adherence junctions were visualized using for 45 min at 37 °C, 5% C O . After the cells attached to a VE-Cadherin stain (Abcam, 33168, diluted 1:1000 in the bottom of the perfusion channel, 50 µL of medium was blocking solution, 30 µL pipetted in the perfusion inlet, added in the perfusion inlet and outlet wells and the plates 20 µL in the perfusion outlet), which was incubated for were placed on an interval rocker platform for continuous 1 h at RT followed by 30-min incubation with Alexa Fluor perfusion. (Perfusion rocker, MIMETAS). The rocker was 488 (ThermoFisher Scientific, A11008, 1:250 in block - set at a 7-degree inclination and 8-min cycle time. Medium ing solution). To perfuse the chips with primary antibody, was refreshed three times a week. the device was placed on a rocker platform. After incuba- tion with the secondary antibody, the device is washed once with washing solution, followed by nuclei staining (NucBlue Fixed cell staining, Life technologies, R37606), and the cytoskeletal marker F-actin, stained by ActinRed™ 1 3 160 Angiogenesis (2019) 22:157–165 555 ReadyProbes® (ThermoFisher Scientific, R37112) in Results PBS and imaged using a high content confocal microscope (Molecular Devices, ImageXpress™ Micro Confocal) at Robust gradient formation in a 3D 10x magnification. microenvironment Sprouting quantification The microfluidic culture platform is based on a 384-well microtiter plate format. The glass bottom contains 40 micro- The average sprouting length was quantified using FIJI v. fluidic units (Fig.  1a), and each microfluidic unit is posi - 1.52 by manual determination of the distance between the tioned underneath nine wells (3 × 3). Every unit consists of microvessel and the tip cell sprouting furthest into the gel. three channels: the center channel that is used to pattern The sprouting length of PMA was obtained after 3 days, an  extracellular matrix (‘gel channel’) and two adjacent all other combinations after 4  days. VEGF + PMA and channels (‘perfusion channels’) (Fig. 1b). The channels are VEGF + S1P microvessels after 6 days of stimulation were separated by PhaseGuides: small ridges that function as cap- used to quantify the median sprout number, average diam- illary pressure barriers, which enable patterning of cells and eter in the minor direction, and circularity. Images were gel without the use of artificial membranes [37]. Every chan- obtained from two replicates for every condition. Using nel has one inlet and one outlet, which connect the channels a 10x objective, we acquired 180 z-steps with 1 µm spac- with the wells in the microtiter plate. Compartmentalization ing and obtained two adjacent sites. The orthogonal views is achieved by patterning a hydrogel in the middle channel were extracted and analyzed in the middle of the gel region. (Fig. 1c, step 1), and enables the formation of gradients by Thresholding of the vessels was automated using Weka Seg- adding a source and sink in the opposite perfusion channels mentation tool [36] (v 3.2.27). Particle analysis was per- (Fig. 1c, step 2). Without continuous replenishment of the formed to include particles between 10 and 10,000 µm with gradients source and sink in the microfluidic channels, gra- a circularity between 0.10 and 1.00. dients 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 continu- ously and simultaneously (Fig. 1c, step 3). As the volume inside the wells is typically orders of a magnitude higher Fig. 1 Gradient generation in a 3D microenvironment. a Bottom of Three-step method to generate gradients in patterned hydrogels. Step the OrganoPlate®, a microfluidic culture platform based on a 384- 1: 2 µL of collagen-1 gel is added in the center channel and polymer- well plate. The glass bottom includes 40 microfluidic devices. b The ized. Step 2: source and sink are added in opposite perfusion chan- geometry of a single microfluidic device that is positioned underneath nels. Step 3: the device is placed on a rocker platform to perfuse both nine wells (3 × 3). Every device consists of three channels: one ‘gel’ perfusion channels continuously to generate a gradient. d Gradient channel for gel patterning, and two adjacent channels. Phaseguides visualization after 1, 3, and 6  days after addition of 20  kDa FITC- prevent the patterned gel from flowing into the adjacent channels. c Dextran as a gradient source 1 3 Angiogenesis (2019) 22:157–165 161 than in the microfluidic channels (the wells typically contain the perfusion channel, while the gel forms the basal side of volumes of 50 µL, compared to < 1 µL in the microfluidic the tube [38]. channels), the source and sink within the microfluidic chan- nels are constant over prolonged periods of time. Thus, a sta- Combination of angiogenic factors is required ble gradient could be maintained for multiple days (Fig. 1d) to induce sprouting without the need to replenish. Although a gradient is still present after 6 days, the steepness is affected due to satura- After reaching confluency in 3  days, the microvessels tion of the sink. Therefore, growth factors and medium were showed a stable morphology of a single monolayer against replaced at 2–3-day time intervals. the gel (Fig. 2b, step 1), despite the numerous (angiogenic) Importantly, the high hydraulic resistance of the hydrogel growth factors that are present in the media (such as vas- limits the influence of differences in hydrostatic pressures. cular endothelial growth factor (VEGF) and basic fibro- This results in a reproducible and robust platform to gener- blast growth factor (bFGF)). We included VEGF and S1P ate gradients, despite the presence of small difference in as they have been shown to induce angiogenic sprouting volumes, for example, due to pipetting errors. Nonetheless, within a collagen-1 matrix [39–41] and included phorbol hydrostatic pressures still can influence the shape of the 12-myristate 13-acetate (PMA) as it has been found to pro- gradient, when the difference between the volumes is suf- mote lumen formation in the absence of fibroblasts [15, 42], ficiently large. This allows different types of gradient to be and used in concentrations of 50 ng/mL for VEGF, 500 nM generated (e.g., linear or parabolic, Supplementary Fig. 1). for S1P, and 2 ng/mL for PMA. The angiogenic growth fac- tor cocktail was added on the basal side of the vessels, and Microvessels cultured against patterned collagen‑1 formed a gradient within the collagen-1 gel (Fig. 2b, step gel 1). This induced the formation of tip and stalk cells after respectively 1 and 2 days (Fig. 2b, step 2–3). After gel loading and polymerization (Fig.  2a, step 1), Interestingly, adding either VEGF, S1P, or PMA alone endothelial cell suspensions were added to the perfusion on the basal side did not result in angiogenic sprouting channels adjacent to the gel. After the cells adhered to the (Supplementary Fig.  2). We quantified the angiogenesis glass substrate (step 2) of the channel, perfusion was applied after addition of various combinations of VEGF, PMA and by placing the device on a rocker platform (step 3). Conflu- S1P (Fig. 3a, b). VEGF + PMA + S1P together  resulted in ent microvessels were formed after 3 days of culture, and the angiogenesis including tip/stalk cell formation, the presence apical side of the vessel (the lumen) can be accessed through of filopodia and lumen formation and directional growth towards the gradient. The sprouts fully traversed the gel after Fig. 2 Microvessel culture against a  patterned collagen-1 gel. a was formed. b Angiogenesis assay using a gradient of angiogenic fac- Method the culture a microvessel within a microfluidic device. First, tors. Angiogenic factors are added once a stable monolayer of ECs is collagen-1 gel is patterned in the middle channel. After polymeriza- formed against the gel (step 1). Addition of a gradient of angiogenic tion, an endothelial cell suspension was added in the adjacent perfu- growth factors resulted in tip cells formation including filopodia at sion channel. By placing the device on a rocker platform, the chan- day 1 (step 2). Lumens formed by the stalk cells are visible at day 2 nels are continuously perfused. After 72  h, a confluent microvessel (step 3) 1 3 162 Angiogenesis (2019) 22:157–165 Fig. 3 Angiogenic sprouts after addition of angiogenic factors. a Stained against F-actin (red) and nucleus (blue) and VE-cadherin Images of sprouting after 4  days of stimulation of a gradient of dif- (green). e Same as c, but stimulation with VEGF + PMA. f Same ferent combinations of angiogenic factors. b Quantification of maxi- as c, but stimulation with VEGF + S1P. g–i Comparison between mum absolute sprouting length in µm after stimulation for 3 (PMA) VEGF + PMA and VEGF + S1P in number of sprouts, diameter, or 4  days (all other combinations) (n = 6). c Angiogenic sprouts and circularity (n = 2). Significance was calculated using one-way after 6 days of stimulation with VEGF + PMA + S1P, stained against anova (b) or Student’s t test (g–i) and shown as n.s (non-significant), F-actin (red) and nucleus (blue). d Close-up of middle (i), top (ii), *(P < 0.05), **(P < 0.01), or ***(P < 0.001). Scale bars: 100  µm. and cross-section (iii) of VEGF + PMA + S1P stimulated sprouting. Graphs are presented as mean ± SD about 6 days and started to form a continuous monolayer towards the gradient compared to VEGF + PMA (Fig. 3i). against in the channel on the other side of the gel and in the Taken together, these results clearly demonstrate that in a basal perfusion channel  (Fig. 3c). The angiogenic sprouts gradient-driven, 3D cell culture environment, a combination have a clear lumen formation (Fig. 3d, panel i), appear cir- of different cues is required to trigger angiogenesis, and S1P cular in a cross-sectional view (Fig. 3d, panel ii), and have is a crucial factor in the distribution and guidance during clear VE-cadherin expression (Fig. 3d, panel iii). angiogenic sprouting. To identify the contribution of PMA and S1P to angio- genic sprouting, we directly compared VEGF + PMA with Anastomosis triggers remodeling and stabilization VEGF + S1P. The combination of VEGF + PMA triggered the formation of angiogenic sprouts into the gel, but the tip Prolonged exposure to growth factors caused the angiogenic cells fail to develop their characteristic tip cell morphology sprouts to anastomose, and connection is formed between the including filopodia and the sprouts lack directionality after two perfusion channels. After anastomosis, we observed a 6 days of sprouting (Fig. 3e and Supplementary Fig. 3a, b). significant reduction of sprouts (Fig.  4a, b). Some angiogenic Furthermore, the sprouts appear to be non-homogenously sprouts display the characteristic steps involved in pruning: distributed within the collagen gel. In contrast, VEGF + S1P first, the lumen collapses, followed by regression of the angio- shows sprouts that are also connected the sprouts to the main genic sprouts towards the parental vessel (Fig. 4a, b, arrows), vessel, but sprouts are equally distributed within the gel with while other angiogenic sprouts remained and increased their a clear directionality towards the gradient (Fig. 3f). Although lumen diameter (Fig. 4a, b arrowheads). there were not significantly more sprouts after VEGF + S1P The formation of perfusable lumen within the sprouts is stimulation (Fig. 3g), the diameter of the sprouts was sig- visualized by perfusion of the main vessel with 0.5 mg/mL nificantly lower (Fig.  3h). We quantified the circularity of 150 kDa TRITC-Dextran (Fig. 4c, d). A surplus of 50 µL is the sprouts to estimate the directionality: a perpendicular added to the inlet well, which fills the parental vessels and sprout appears circular in a cross-sectional view with a value flows into the angiogenic sprouts. When angiogenic sprouts closer to 1, while a deviating sprout appears flattened (closer did not connect to the basal perfusion channel (Fig. 4c), spots to 0). This shows that VEGF + S1P sprouts have a signifi - were visible within the collagen where dextran leaks out of the cantly higher circularity and thus improved directionality tip of the sprouts (panel ii, left, 0 min). These spots increased 1 3 Angiogenesis (2019) 22:157–165 163 Fig. 4 Anastomosis with basal channel triggers pruning and matura- obtained every minute and directly after addition of a 0.5  mg/mL tion of angiogenic sprouts. a Angiogenic sprouts 5  days after addi- 150 kDa TRITC-Dextran solution in culture media. Panel ii shows the tion of VEGF + PMA + S1P. Compared to the angiogenic sprouts at pseudo-colored fluorescent images after 0 and 9 min after addition of day 8. b Some sprouts regressed (arrows) while other sprouts remain the dextran solutions. Time is indicated in min. d Same as in c, but and showed increased lumen diameter (arrowheads). c Angiogenic after 9  days of stimulation. Sprouts are connected to the other side sprouts after 4 days of stimulation invaded into the gel but are not yet and formed a confluent microvessel in the basal perfusion channel. connected to the bottom perfusion channel. Fluorescent images were Scale bars: 100 µm over time (right, 9 min). However, after anastomosis (Fig. 4d), important mechano-biological signal in during angiogen- sprouts retained the dextran in their lumen, and shows subse- esis [43], while flow in this assay occurs at discrete time quent l fi ling of the bottom basal perfusion channel. This shows points and is bi-directional. Thus, despite the evidence that that sprouts stabilize and form a functional barrier after a con- flow affects the remodeling and maturation of the capil - nection has been formed. laries in our model, the exact contribution of flow in this assay is difficult to determine. We showed that gradient-driven angiogenic sprouting Discussion through an extracellular matrix requires not just the pres- ence of VEGF, but a combination of multiple angiogenic We report a robust, standardized microfluidic cell cul- factors [44]. The combination of VEGF + PMA + S1P was ture platform to study gradient-driven angiogenesis of the most optimal cocktail to trigger quick, robust, directional a perfused microvessel in high-throughput. Each device angiogenesis with angiogenic sprouts with clear lumen for- contains 40 individually addressable microfluidic units mation. VEGF + PMA showed a random distribution of the and enables the culture of 40 identical microvessels. An sprouts and an absence of filopodia on the tips cells, and important advantage of this assay is the defined geometry the sprouts lacked directionality. In contrast, a VEGF + S1P of the microfluidic channels, as this results in reproducible gradient showed formation of angiogenic sprouts, includ- experimental cell culture conditions (position and density ing tip cells with filopodia. Filopodia allow the tip cells to of the cells, amount of flow, position of the extracellular sense a biochemical gradient [4], and explains the observed matrix and the shape of the gradient) and increases the directionality of the angiogenic sprouts. This suggests that robustness and scalability of our assay. S1P plays an important role in the differentiation into func- Perfusion in our device is induced by passive leveling tional tip cells and the observed repetitive formation of angi- using a rocker platform, and has two crucial advantages. ogenic sprouts. Such a repetitive formation of angiogenic First, the flow is simultaneously applied throughout all sprouts can be explained by a reaction–diffusion mecha- microfluidic units, which results in reproducible gradient nism between VEGF and Flt-1, the soluble form of VEGF formation. Second, as tubing and pumps are not required receptor. Stalk cells are known to secrete Flt-1, which binds the throughput is greatly increased: the assay is scalable VEGF and prevents neighboring cells to become tip cells since multiple experiments can be performed by stacking [45]. This is required for efficient angiogenic sprouting into of culture platforms on top of each other. Nonetheless, the matrix [3], with evenly distributed sprouts roughly every using a rocker platform to induce flow is also a trade- 100 µm, as predicted in silico [8, 9]. It has been shown that off that has its downsides: first, the requirement of a S1P has a pro-angiogenic effect in vitro [39, 40, 46–48] and rocker platform limits us to perform time-lapse imaging in vivo [39, 49, 50]. Our data suggest a pro-angiogenic syn- only at discrete time points, as the vessels and gradient ergy between S1P and VEGF, which is in agreement with require continuous perfusion. Second, vasculature in vivo the fact that inhibition of S1P also prevents VEGF-induced is exposed to continuous, unidirectional flow that is an angiogenesis in vivo [51]. Interestingly, S1P is also known 1 3 164 Angiogenesis (2019) 22:157–165 for its barrier stabilizing, anti-angiogenic properties, and Conclusion vascular maturation [52, 53]. Therefore, we hypothesize that the effect of S1P is dependent on whether it is present We demonstrate a gradient-driven, three-dimensional angio- on the apical side of ECs (lumen) or basal side, either medi- genesis assay in a standardized microfluidic platform. Angi- ated by differences in apical and basal expression of S1P ogenic sprouting is induced from a perfused microvessel receptors [54] or by dimerization with other receptors, like through a patterned collagen-1 gel. The combination of angi- basally expressed VEGFR2 [46]. A better understanding of ogenic factors was optimized to trigger angiogenic sprout- the precise mechanisms of S1P signaling in angiogenesis ing that faithfully reproduces all the angiogenic events that will provide therapeutic strategies that specifically target the occur in vivo, such as the differentiation of the endothelial pro-angiogenic effects of S1P [49]. cells into tip, stalk, and phalanx cells and the formation of Prolonged exposure (> 6 days) to a gradient of angio- perfusable lumen. It was found that a combination of VEGF, genic stimuli resulted in sprouts that connect the two perfu- S1P, and PMA provided the optimal cocktail for 3D angio- sion channels (anastomosis). This connection resolves the genic sprouting. After the angiogenic sprouts anastomosed gradient, as there is a direct connection between the source through the collagen to the other channel, remodeling and and sink, and also results in the onset of flow through the stabilization of the capillary bed was observed. sprouts. There remains controversy about the exact mecha- Acknowledgements We thank Angiocrine for the kind gift of VeraVec nism that leads to pruning. In vivo, this is either shear-medi- HUVEC cells. V. van Duinen was partially financially supported by ated or due to changing receptor expression after a resolved the STW Valorisation Grant (STW 12615), the VIRGO consortium (oxygen) gradient [10, 55]. Once anastomosis occurred, we (FES0908), and supported bythe Dutch Heart Foundation (CVON observed remodeling of the capillary bed, including pruning RECONNECT) and ZonMw (MKMD:114022501) Grant to T. Hanke- meier and A. J. van Zonneveld. and regression of angiogenic sprouts within the collagen. Furthermore, some sprouts increased in lumen diameter, Author contributions VD, CR and DZ performed the experiments. VD likely caused by the onset of perfusion [56]. By controlling wrote the manuscript with input from all authors. PV, TH and AJZ shear levels and oxygen tension in this assay, we will be able supervised all aspects of the work. to determine which of those effects is the crucial mechanism in pruning. Compliance with ethical standards Perfusion of the sprouts with fluorescently labeled dex- tran showed that angiogenic sprouts that did anastomose are Conflict of interest P. Vulto and T. Hankemeier are shareholders in Mimetas BV. V. van Duinen, D. Zhu, C. Ramakers and A. J. van Zonn- permeable near the tip/stalk cell region. In contrast, anasto- eveld declare no potential conflict of interest. mosed sprouts retained the 150 kDa dextran solution within their lumen, suggesting that the connection between the two Open Access This article is distributed under the terms of the Crea- channels triggers maturation of the ECs in the sprouts, as tive Commons Attribution 4.0 International License (http://creat iveco they adopt their characteristic phalanx phenotype including mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate mature cell–cell junctions [55, 57]. Furthermore, once the credit to the original author(s) and the source, provide a link to the angiogenic sprouts connected, the medium can be switched Creative Commons license, and indicate if changes were made. back to the original culture medium with low levels or growth factors, while the integrity of the sprouts remained (Supplementary movies 2, 3), which suggests that perfusion is an important survival factor for angiogenic sprouts in the References absence of a high concentration of angiogenic factors like VEGF. 1. Carmeliet P (2005) Angiogenesis in life, disease and medicine. We expect that our platform will be widely adopted for a Nature 438(7070):932–936 2. Carmeliet P, Jain RK (2000) Angiogenesis in cancer and other range of applications, including both fundamental studies of diseases. Nature 407(6801):249–257 the mechanisms of angiogenesis as well as for the identic fi a - 3. 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Journal

Angiogenesis – Springer Journals

Published: Aug 31, 2018

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