The physiological relevance of Matrigel as a cell-culture substrate and in angiogenesis assays is often called into question. Here, we describe an array-based method for the identification of synthetic hydrogels that promote the formation of robust in vitro vascular networks for the detection of putative vascular disruptors and that support human embryonic stem cell expansion and pluripotency. We identified hydrogel substrates that promote endothelial-network formation by primary human umbilical vein endothelial cells and by endothelial cells derived from human-induced pluripotent stem cells, and used the hydrogels with endothelial networks to identify angiogenesis inhibitors. The synthetic hydrogels showed superior sensitivity and reproducibility over Matrigel when known inhibitors were evaluated, as well as in a blinded screen of a subset of 38 chemicals, selected according to predicted vascular disruption potential, from the Toxicity ForeCaster library of the United States Environmental Protection Agency. We propose that the identified synthetic hydrogels are suitable alternatives to Matrigel for common cell-culture applications.
Owing to the increasing number of diseases associated with vascular disorders, the ability to detect compounds that affect the human vasculature is becoming more important. Vascular disorders include various forms of cancer, atherosclerosis, stroke, diabetic retinopathy and developmental complications, all of which can result from exposure to certain chemicals present in the environment1,2. In this regard, early studies led to the development of an in vitro endothelial-network-formation assay3, in which cloned capillary endothelial cells formed interconnected endothelial networks after approximately 10 days in culture. A more rapid assay (<24 h) described in 1988 has become a gold-standard method for the identification of inhibitors and stimulators of angiogenesis in drug discovery4 and toxicological screening2,5,6,7,8,9.
The assay developed in 1988 has a number of inherent complexities, due in part to the use of a natural extracellular matrix (ECM) derived from Engelbreth–Holm–Swarm tumours produced in mice and referred to as Matrigel, Engelbreth–Holm–Swarm matrix or Geltrex (hitherto referred to as Matrigel)10. Matrigel is used in multiple applications as a substrate in human cell culture and organoid assembly. Two of its most common uses are in angiogenesis assays and in the expansion of undifferentiated human embryonic stem cells (hESCs)11,12. However, Matrigel is inherently limited by its compositional complexity and lack of lot-to-lot reproducibility. Recent proteomic analysis of normal and ‘growth-factor reduced’ Matrigel identified over 1,500 unique proteins, and individual tests from two manufacturers showed only 53% batch-to-batch similarity in the proteins identified13. The low and variable elastic modulus of Matrigel, which ranges from of 0.12 to 0.45 kPa (ref. 14), results in poor handling characteristics, the need for precise temperature control and user-to-user variability. Numerous confounding factors, such as locally sequestered and matrix-bound growth factors15, as well as physiologically irrelevant mechanisms of inhibition, such as bulk matrix dissolution by Suramin treatment16,17, have previously resulted in the identification of false positives and false negatives in Matrigel-based chemical compound screenings. Moreover, the introduction of xenogenic components by Matrigel interferes with mechanistic studies of cell behaviour and limits therapeutic applications of stem cells expanded in culture12.
Synthetic and natural ECMs—for example, collagen, fibrin and vitronectin—are suitable alternatives to Matrigel for assembling endothelial networks and for expanding stem cell populations. Natural ECMs, however, are often derived from animals (bringing xenogenic material into the culture environment) and are often presented as coatings that fail to mimic the mechanical properties of the native ECM. In endothelial-network-formation assays, other natural ECMs fail to form endothelial networks similar to those of Matrigel without the addition of a supporting cell type. Chemically defined synthetic hydrogels have received increased attention as suitable alternatives to Matrigel and other natural ECMs due to their minimal batch-to-batch variation, increased reproducibility, defined material properties, defined compositions and controllable degradation properties18,19,20. Isolated components of Matrigel, such as laminin and collagen type IV, and synthetic poly(ethylene glycol) (PEG) hydrogels in the form of PEG-vinyl sulfone (PEG-VS) enzymatically crosslinked with varying stiffness were systematically screened to control early events in neurogenesis. hESCs were encapsulated in a broad range of hydrogel conditions. Material properties were shown to impact their differentiation towards an ectodermal fate and their later dorsoventral patterning was shown to model that of the developing hindbrain and spinal cord21. Another study demonstrated the ability of synthetic PEG hydrogels to optimize the reprogramming efficiency of mouse and human fibroblasts into induced pluripotent stem cells and later maintain their phenotype in three-dimensional environments22. It has also been demonstrated that chemically defined synthetic hydrogels have the ability to support the expansion of intestinal stem cells and the formation of intestinal organoids23, control the formation of epithelial Madin–Darby kidney cysts24, alter stem cell fate 25,26,27,28,29 and generate tumourigenesis models30,31,32,33 that are similar or superior to Matrigel and their natural ECM counterparts.
In this study, we applied an array-based method of optimizing synthetic hydrogels for use in vascular toxicity assays and hESC expansion. We used a synthetic hydrogel composed of photo-crosslinked, cell-degradable PEG hydrogels formed via a step-growth reaction to generate hydrogels with controlled mechanical properties and presentation of bioactive peptides that mimic functional groups of larger ECM molecules. The chemistry used in this study, comparisons with other materials and array-based methods are discussed in the Supplementary Information. We evaluated over 1,200 distinct synthetic hydrogels and identified chemically defined, synthetic hydrogels that replace the role of Matrigel in vascular screening assays and hESC expansion. The resulting PEG hydrogels exhibit defined characteristics, including cell adhesion properties, shear modulus (stiffness) and non-covalent binding affinity for vascular endothelial growth factor (VEGF). These hydrogels support the robust formation of endothelial networks from endothelial cells derived from multiple primary human sources, and exhibit unprecedented batch-to-batch reproducibility and increased sensitivity to known angiogenesis inhibitors. The synthetic hydrogel-based assay was superior to the commonly used Matrigel-based assay in its ability to qualify the relative anti-angiogenic activities across a 38-chemical subset from the United States Environmental Protection Agency (EPA)’s ToxCast chemical library, which was selected for evaluation based on in vitro cellular and molecular bioactivity profiles that predict their potential for vascular disruption, ranging from inactivity to strong inhibition. Similarly, the array-based hydrogel identification methods applied in these studies enabled the discovery of suitable substrates for hESC adhesion and maintenance of pluripotency. These results indicate that an array-based discovery approach can be used to identify relatively simple synthetic materials that provide superior utility when compared with more complex naturally derived materials.
Approach for the identification of synthetic hydrogel formulations
The approach for the identification of synthetic hydrogels that supported specific intended cell behaviours (for example, tubulogenesis and hESC culture) used arrays of 120-μm-thick synthetic hydrogels (thin hydrogel arrays) that mimicked the properties of the native ECM. The inclusion of pendant linear H–Cys–Arg–Gly–Asp–Ser–NH2 (linear RGD) or head-to-tail cyclized Arg–Gly–Asp–[d-Phe]–Cys (cyclic RGD) peptides mediated cell adhesion to the hydrogels through the RGD motif commonly present in integrin-binding ECM proteins, such as laminin, vitronectin and fibronectin34 (Fig. 1a,d). The mechanical properties of the hydrogels were set at 0.45 ± 0.04 kPa (soft), 1.16 ± 0.08 kPa (medium) or 4.72 ± 0.17 kPa (stiff) by tuning concentrations of 20 kDa, eight-arm PEG-norbornene (PEGNB) and dithiol-terminated crosslinking molecules used to form the hydrogels (Fig. 1d and Supplementary Fig. 1a). All hydrogel solutions were polymerized using a crosslinking peptide H–Lys–Cys–Gly–Gly–Pro–Gln–Gly–Ile–Trp–Gly–Gln–Gly–Cys–Lys–NH2 that was degradable by matrix metalloproteinases (MMPs)35, thereby enabling cell-mediated remodelling of the hydrogel substrates36. All peptides included in the hydrogels were covalently attached to PEG via thiol-ene ‘click’ chemistry, which couples thiols to norbornene groups on the PEG molecules37. Finally, the ability to bind and sequester VEGF was provided by a receptor-mimicking VEGF-binding peptide (VBP) included in the hydrogels38 (Fig. 1a and Supplementary Fig. 2). Further methods and results for evaluating hydrogel crosslinking efficiency and long-term swelling behaviour are in the Supplementary Information. Taken together, these hydrogels presented a simple, chemically defined set of cell-signalling cues relative to the complex ECM presented by Matrigel.
Identification of materials to promote endothelial-network formation
Thin hydrogel arrays (Fig. 1b) identified cell-culture environments that supported robust endothelial-network formation by human umbilical vein endothelial cells (HUVECs) as well as endothelial cells derived from human-induced pluripotent stem cells (iPSC-ECs). Successful culture environments promoted endothelial-network assembly by endothelial cells within 24 h of seeding (Fig. 1c) and maintained network stability 48 h after seeding (Fig. 1d). Multiple culture environments successfully supported HUVEC network formation after 24 h, but HUVEC networks typically disassembled during the subsequent 24 h period. A single condition, characterized as 2 mM PEG, 0.125 mM cyclic RGD and 4 mM crosslinking peptide (50% crosslinked) in the substrate, with 5 ng ml–1 soluble VEGF present in the media, supported prolonged endothelial-network stability for 24 h after initial endothelial-network formation (Fig. 1d). Interestingly, the 0.45 kPa stiffness of the identified hydrogel fell approximately within the average stiffness range of Matrigel14. As demonstrated during time-lapse microscopy, the synthetic hydrogel formulation identified here reproducibly formed endothelial networks within 6–8 h of seeding (Fig. 1c and Supplementary Video 1). Additionally, the identified hydrogel formulation was suitable for embedding substrates in aortic ring sprouting assays (Supplementary Fig. 5). Further discussion of this experiment is in the Supplementary Information.
Thin hydrogel arrays also identified culture conditions that promoted endothelial-network assembly by iPSC-ECs39. Multiple culture environments supported iPSC-EC network formation within 24 h after seeding and maintained network stability for 24 h after the initial endothelial-network formation (Fig. 1d). Successful culture environments detected by the thin-hydrogel arrays were adapted for use in 96-well plates for toxicity screening experiments. The amount of photoinitiator and volume of hydrogel used were optimized to 0.2% w/v Irgacure 2959 (I2959) and 9 μl solution per well, respectively, to produce hydrogels with the flattest and most uniform surfaces for the seeding of HUVECs or iPSC-ECs (Supplementary Fig. 6) without changing the fundamental swelling properties of the hydrogels (Supplementary Fig. 4c). This reduced hydrogel buckling and out-of-plane imaging of the 96-well plate, resulting in reduced imaging time and non-uniformity between samples.
Confocal images showed the organization of HUVECs and iPSC-ECs into clearly branching, interconnected networks on both synthetic hydrogels and Matrigel (Figs 1d and 2a). All endothelial cells forming networks on synthetic hydrogels displayed an ability to migrate and develop cell–cell contacts consistent with the process of capillary morphogenesis in vivo (Fig. 1c and Supplementary Video 1–Supplementary Video 3). The ability to form endothelial networks on the synthetic hydrogels was not limited to endothelial cells expanded in culture. Similar endothelial networks were formed directly from cryopreservation (Supplementary Fig. 1b,c).
Utility of synthetic hydrogels for vascular inhibition assays
Synthetic hydrogel-based endothelial-network formation assays accurately and reproducibly distinguished endothelial networks disrupted using the known angiogenesis inhibitor Sutent from non-inhibited networks. Quantification of the total endothelial network area in the inhibited and non-inhibited conditions (Fig. 2b,c) resulted in a Z′ score of 0.66 for the synthetic hydrogel-based assay (considered an ‘excellent’ assay40), while HUVEC networks formed on Matrigel resulted in a Z′ score of −0.74, indicating that inconsistencies in the Matrigel-based assay interfere with the identification of inhibiting versus non-inhibiting conditions (Fig. 2d). Similarly, screening for Sutent activity on iPSC-EC networks formed on synthetic hydrogels produced a favourable Z′ score of 0.20, while iPSC-EC networks formed on Matrigel resulted in a poor Z′ score of −1.06 (Fig. 2d).
HUVEC network sensitivity to known VEGF signalling inhibitors
HUVEC networks on synthetic hydrogels showed increased sensitivity to a number of known VEGF inhibitors compared with HUVEC networks on Matrigel, including Vatalanib, Semaxanib, Sutent and a soluble fms-like tyrosine kinase 1 (sFlt-1) receptor (Figs 3 and 4 and Supplementary Fig. 7a). In all of the above cases, the VEGF inhibitors significantly inhibited network formation in a wider range of concentrations in the synthetic hydrogel-based assay compared with the Matrigel-based assay. Vatalanib showed inhibition at 2.5–20 μM concentrations on synthetic hydrogels, while inhibition was not detected on Matrigel. Semaxanib showed inhibition at concentrations of 0.63 μM and 10–20 μM on synthetic hydrogels, while inhibition was not detected on Matrigel. Anti-VEGF did not show inhibition on synthetic hydrogels or Matrigel. Sutent showed inhibition at concentrations of 1.25–20 μM on the synthetic hydrogels, while inhibition was detected at a concentration of 0.31 μM on Matrigel. On Matrigel, this effective concentration was subject to change significantly across technical replicates. Comparisons of these results with those demonstrated in the literature may be found in the Supplementary Information.
To gain insight into the influence of VEGF sequestering on the performance of the tubulogenesis assays, we generated a separate set of synthetic hydrogels that supported endothelial-network formation (Supplementary Fig. 2), but also included VBP. Interestingly, the presence of VBP in the synthetic hydrogels increased the effectiveness of the VEGF receptor tyrosine kinase inhibitors Vatalanib and Sutent in disrupting HUVEC network formation. In contrast, sFlt-1, which binds soluble VEGF in media, did not result in significant changes to the network area at any of the tested concentrations when the synthetic hydrogel contained VBP. The effective concentration ranges of Semaxanib and Prinomastat hydrochloride, a broad-based inhibitor of MMP-2, -3, -9, -13 and -14, were no different between the hydrogels that contained or lacked VBP. These data indicate that VEGF sequestration changes the sensitivity of HUVEC networks to VEGF inhibitors, depending on whether they affect receptor tyrosine kinase activity or the activity of soluble VEGF as possible mechanisms of inhibition (Figs 3 and 4 and Supplementary Fig. 8).
A differential response to MMP inhibition and known Matrigel dissolution agents
HUVEC networks on synthetic hydrogels were more sensitive to an MMP inhibitor than HUVEC networks on Matrigel. HUVEC network formation on each of the synthetic hydrogels—with or without VBP—was inhibited by Prinomastat hydrochloride and resulted in confluent cell-sheet formation rather than network formation. In contrast, HUVEC network formation on Matrigel was unaffected by this MMP inhibitor (Fig. 4 and Supplementary Figs 7b and 9). Interestingly, the presence of VBP in the synthetic hydrogels did not interfere with the inhibitory activity of Prinomastat hydrochloride, and treatment also resulted in cell-sheet formation rather than network formation. This is consistent with the expectation that the actions of Prinomastat hydrochloride are independent of VEGF signalling (Figs 3 and 4).
HUVEC networks on synthetic hydrogels were unaffected by a putative matrix dissolution agent, which dramatically disrupted network formation on Matrigel. Suramin hydrochloride, an inhibitor to epidermal growth factor, platelet-derived growth factor and transforming growth factor-β signalling16, inhibited network formation on Matrigel in a dose-dependent manner, and resulted in cell clustering indicative of apparent Matrigel dissolution17 and cell settling on an underlying polystyrene cell-culture substrate. On the synthetic hydrogels, suramin hydrochloride treatment minimally affected HUVEC network formation and did not dissolve the underlying material, suggesting that the inhibitory effect on Matrigel was attributable to substrate dissolution rather than vascular inhibition (Supplementary Fig. 10), as demonstrated previously17. This result illustrates a concern with the interpretation of results from Matrigel endothelial-network-formation assays, as Matrigel dissolution is not a process relevant to disorders of in vivo angiogenesis.
Identification of putative vascular disrupting chemicals from the ToxCast compound library
As a further investigation of the enhanced utility of the synthetic hydrogel system, we ran a comparison utilizing a subset of candidate chemical compounds from the ToxCast library. This test set included 38 unique chemicals ranging in their putative vascular-disrupting chemical (pVDC) scores based on a signature derived from the ToxCast phase I screen of 309 chemicals screened across 500 high-throughput screening assays41,42. The ToxCast signature was translated by a pVDC cutoff provisionally set to 0.100, wherein 26 chemicals were predicted to have some anti-angiogenic activity and 12 chemicals were predicted to be inactive1,41,43,44,45, 46,47. To best qualify this evaluation, (1) the experimenters were blinded to the test panel, and (2) the test panel included 38 unique chemicals and an assortment of replicates to yield 53 samples. Each of these 53 samples was run in triplicate. The results for the positive and negative calls after 24 h in culture are shown for the blinded sample matrix (Fig. 5) and for the decoded list (Table 1). The synthetic hydrogel assay confirmed 11 out of the 26 provisional positives, while the Matrigel assay detected only five. An overlap of four was detected by both platforms (Fig. 5, Supplementary Fig. 11 and Table 1). In both cases, all positive calls had higher pVDC scores (>0.1), predicting greater disruptive activity (Table 1). Both the synthetic hydrogel and Matrigel assays identified an increasing number of inhibitors as pVDC scores increased. Inhibitory ‘hit’ conditions and non-inhibitory ‘miss’ conditions were compared with predicted inhibitory or non-inhibitory calls in a confusion matrix anchored to the pVDC domain (Tables 1 and 2; ref. 48). The test yielded an accuracy value of 60.5% for the synthetic hydrogel assay and 44.7% for the Matrigel assay. The sensitivities were 42.3% and 19.2% for the synthetic hydrogel and Matrigel assays, respectively, and the specificity in both systems was 100%. An F1 score, which measures the harmonic mean of precision and recall, was 0.59 in the synthetic assay and 0.32 in the Matrigel assay (scored from 0 to 1). Matthew’s correlation coefficient, which measures the quality of binary classification, was 0.43 in the synthetic hydrogel versus 0.26 in the Matrigel assay (scored from 0 to 1). The balanced accuracy, which takes into account both the sensitivity and specificity of both systems and removes potential bias from unbalanced datasets49, gave a value of 72.2% for the synthetic assay versus 68.1% for the Matrigel assay.
Identifying hydrogels to maintain hESC pluripotency during initial expansion
Thin hydrogel arrays (Fig. 1b) identified multiple hydrogel formulations that maintained short-term NANOG expression by hESCs at greater or equal levels compared with hESCs cultured on Matrigel, four days after seeding (Fig. 6a). Defined environmental properties included three varying levels of hydrogel stiffness—1.5 kPa (soft), 3 kPa (medium) and 10 kPa (stiff)—(Fig. 6 and Supplementary Fig. 12), a range of cyclic RGD cell adhesion peptide concentrations in the hydrogels and rho-associated protein kinase (ROCK) inhibitor (Y-27632) concentrations in the medium during seeding and during the four-day maintenance period. From a total of 64 possible conditions (Fig. 6), a number of hydrogel formulations successfully maintained NANOG expression to levels greater than or equal to the Matrigel controls (Fig. 6a). One hydrogel formulation in particular, which contained 4 mM PEG, 2 mM cyclic RGD and 12 mM 3.4 kDa dithiolated PEG crosslinking molecule (10 kPa), increased NANOG expression without the need to include the ROCK inhibitor in either the cell seeding or during maintenance on the hydrogel substrate. Other conditions, which contained 4 mM PEG, 4 mM cyclic RGD and 12 mM 3.4 kDa dithiolated PEG crosslinking molecule (10 kPa), enabled increased NANOG expression along with increased cell adhesion relative to Matrigel (Fig. 6). These conditions encompassed situations in which hESCs were seeded as single cells or colonies, with or without ROCK inhibitor during culture maintenance.
When seeded on synthetic hydrogels, the hESCs that expressed elevated levels of NANOG also expressed elevated levels of octamer-binding transcription factor (OCT)3/4 and sex determining region Y-box 2 (SOX)-2 compared with the hESCs on Matrigel. Following the completion of the initial screen, hESCs were seeded as single cells onto a subset of hydrogels containing 1 to 4 mM cyclic RGD with stiffnesses of either 3 or 10 kPa. The 3 kPa hydrogels containing 1 and 2 mM cyclic RGD increased NANOG expression by hESCs compared with hESCs on Matrigel (Fig. 6 and Supplementary Fig. 13), while all other hydrogels in the tested range of conditions decreased NANOG expression. On most hydrogels in this range of conditions, hESCs expressed elevated OCT3/4 and SOX-2 relative to Matrigel (Supplementary Fig. 13). This change in marker expression was detectable using NANOG as a marker of pluripotency, but was not detectable using OCT3/4 or SOX-2.
Through the use of hydrogel arrays, we identified alternative synthetic substrates to Matrigel for use in angiogenesis assays (Figs 1-5) and short-term hESC expansion (Fig. 6). In angiogenesis assays, synthetic hydrogels mediated endothelial-network formation by HUVECs and iPSC-ECs, enabled the evaluation of known pharmacological inhibitors of angiogenesis at concentrations previously demonstrated as effective in vitro50,51,52,53 and enabled the accurate detection of blinded vascular-disrupting chemicals. We demonstrated distinct advantages of synthetic hydrogel-based assays over Matrigel-based assays in terms of reproducibility, as suggested by Z′ scores, and in accuracy, as suggested by the blinded 38 compound screen. The rationale behind the use of thiol-ene photo-crosslinking chemistry for hydrogel crosslinking and the optimization of hydrogel properties is in the Supplementary Information. The screening techniques described here are expected to enable the discovery of hydrogels that mediate network formation by a variety of endothelial cell types beyond those used in these studies, including tissue-specific human endothelial cells and endothelial cells from non-human species. In hESC expansion, cells seeded on synthetic hydrogels expressed greater or equal levels of the pluripotency markers NANOG, OCT3/4 and SOX-2 compared with cells seeded on Matrigel. These outcomes can be attributed to the fact that synthetic hydrogels provided defined levels of adhesion molecule concentration, substrate stiffness and growth factor sequestration to control cell behaviour, while minimizing the variable and unwanted signalling that is normally present in poorly defined materials such as Matrigel. This supports previous studies, the first of which showed that the encapsulation of hESCs in a synthetic PEG hydrogel demonstrates enhanced reprogramming efficiency and maintenance of pluripotency in three dimensions22, and the second of which showed that controlling material parameters using array-based methods could affect stem cell pluripotency and colony formation from individual mouse embryonic stem cells29.
The physiological relevance of Matrigel as a cell-culture substrate in an angiogenesis assay is often called into question as multiple non-endothelial cell types, including melanoma, glioblastoma, numerous breast cancer cell lines, retinal epithelial cells, lens cells, murine Leydeg cells and human fibroblasts54,55,56,57, have formed cellular networks resembling vasculature while on Matrigel. It has been suggested that these networks form via cell traction and malleability of the matrix57,58 rather than relevant mechanisms of vascular morphogenesis, such as the single-cell migration demonstrated by HUVECs on synthetic hydrogels (Fig. 1c and Supplementary Video 1 and Supplementary Video 2; ref. 59). The process by which cancer cells form vascular networks has been viewed as being similar to an in vivo process dubbed vasculogenic mimicry60, and processes such as these likely generate misleading insights into vascular-network formation and false negatives when screening for vascular-disrupting chemicals. For example, we suggest that the differential processes of morphogenesis on synthetic hydrogels and Matrigel impact endothelial cell morphologies, which may distinguish between various mechanisms of vascular inhibition. We demonstrated that on synthetic hydrogels: (1) inhibitors to VEGF receptor binding (for example, sflt-1) induced detectable network broadening over elongation; (2) inhibitors to receptor tyrosine kinase activity (for example, Sutent) disrupted cell–cell contacts and induced the onset of rounded HUVEC morphologies; and (3) inhibitors to MMP activity (for example, Prinomastat hydrochloride) induced the formation of confluent HUVEC monolayers rather than networks (Supplementary Figs 7 and 9). However, while these morphological outcomes were identifiable on a defined synthetic substrate, they were masked if similar assays were performed on Matrigel (Fig. 3 and Supplementary Figs 7 and 9–11). This can be attributed to interfering, poorly defined signals provided by Matrigel, as well as the aforementioned contraction mechanism used by cells on Matrigel. One such source of interference is the binding and sequestration of growth factors, including VEGF, by Matrigel. A discussion of this mechanism may be found in the Supplementary Information.
The goal of the synthetic hydrogel and Matrigel-based angiogenesis assays was to leverage enough endothelial cell functionality, including adhesion, migration and the establishment of cell–cell contacts, to generate endothelial networks as a functional readout of pVDC activity. We did not expect to recapitulate other aspects of endothelial cell functionality, such as MMP-mediated angiogenic sprouting46, in these assays due the use of two-dimensional surfaces as culture environments. Interestingly, our studies suggest that network formation on synthetic hydrogels still requires MMP activity, as MMP inhibition by Prinomastat hydrochloride resulted in monolayer formation rather than network formation (Fig. 3 and Supplementary Figs 7 and 9). Although the synthetic hydrogels used in these studies were MMP labile, endothelial-network assembly has been documented on substrates that are not MMP labile61. Therefore, it is unlikely that MMP-driven substrate degradation was the sole process driving network formation on the synthetic substrates here. We expect that MMP activity mediates other cellular functions during network formation on synthetic hydrogels, including the functions of integrins, growth factor receptors and vascular endothelial cadherin62. While future studies are necessary to characterize the roles of MMP in the context of network formation, the synthetic hydrogels are well suited for these studies as MMP inhibition was difficult to detect on Matrigel (Fig. 3 and Supplementary Figs 7 and 9).
Synthetic hydrogel and Matrigel-based angiogenesis assays identified putative vascular-disrupting chemicals in a blinded subset of 38 chemical compounds taken from the EPA’s ToxCast library. The results from this experiment contributed to an ongoing effort by the EPA to identify potential developmental neurotoxins, and the significance of this work is highlighted in an Organization for Economic Cooperation and Development adverse outcome pathway (OECD AOP43; https://aopwiki.org/aops/43). Both assays were more likely to positively identify candidate positives if their pVDC scores were high, and neither assay of the high-scoring compounds as disrupting chemicals (Fig. 5). As only a single concentration was tested here (10 μM), versus a range of test concentrations comparable to the high levels in ToxCast that generated the pVDC list, we hypothesize that the sensitivity of both assays might be enhanced by increasing the test concentrations. For example, thalidomide (5HPP-33), which is known to inhibit cell proliferation and migration63, was not detected by the synthetic hydrogel assay here. In cases like these, some inhibitors were clearly beginning to show effects of destabilizing endothelial networks (Supplementary Fig. 11b), and a higher concentration of inhibitor may have sufficiently changed the network area to enable the detection of inhibition. Alternatively, the angiogenesis assays here focused only on network formation and did not encompass the full range of cell functions exhibited in angiogenesis, whereas the ToxCast predictions are based on knowledge about the full range of the angiogenic cycle, including vasculogenesis, angiogenesis and angioadaptation. For example, O-(chloroacetyl-carbamoyl) fumagillol (TNP-470), a clinical anti-angiogenesis compound that reduces the permeability and dilation of mature blood vessels while not necessarily affecting developing vessels64,65, was not detected by either assay here. We expect that mechanisms such as these would not have a detectable impact on cell activity in a single functional angiogenesis assay. The pVDC scores of the ToxCast compounds were originally derived from numerous in vitro technology platforms, literature searches and computational methods to measure direct chemical interactions with cell receptors and the resulting changes in gene expression levels1,41,43,44,45,46,47 . However, caution should be exercised when using a high pVDC score as an absolute assurance of disruptive activity, and emerging functional angiogenesis assays may identify high-scoring chemicals as false positive inhibitors. As our assay and other functional angiogenesis assays46 emerge and highlight varying endothelial-cell functionalities in angiogenesis, pVDC scores should be revised over time.
To explore the versatility of the screening methods described here, synthetic hydrogels were customized to replace the ability of Matrigel to maintain short-term hESC pluripotency. Hydrogel arrays identified multiple culture conditions that maintained hESC NANOG expression to greater or equal levels measured on equivalent Matrigel substrates, and these conditions spanned a wide parameter field (Fig. 6a). It should be noted that NANOG was initially used as the marker for pluripotency as, compared with OCT3/4, SOX2 and C-myc, it is the first pluripotency marker lost on differentiation 66,67,68 . Over the course of the pluripotency evaluation described here, NANOG, OCT3/4 and SOX-2 were highly co-expressed in pluripotent hESCs, but in multiple environments hESCs expressed elevated OCT3/4 and SOX-2 but not elevated NANOG compared with Matrigel, and we expect this to signify differentiation (Supplementary Fig. 13). These initial discoveries will enable future explorations of how multiple cell-culture variables, such as single-cell seeding and colony seeding techniques, affect long-term hESC expansion. Additionally, as the well-defined nature of the synthetic hydrogels enabled control of cell morphology during expansion, hESC expansions may be performed in environments that promote either low or high cell adhesion (Fig. 6b). Notably, the hydrogel arrays detected a synthetic hydrogel substrate that increased NANOG expression without the inclusion of ROCK inhibitor Y-27632 in media during cell seeding or maintenance. Previous studies have used synthetic cell-culture substrates for the long-term expansion of pluripotent stem cells, but these procedures required the use of ROCK inhibitor69,70, which has poorly understood effects on long-term hESC expansion71. Additionally, previous studies were not always performed using hESCs. Instead, mouse embryonic stem cells, which may demonstrate different responses to underlying material properties, have been used29. The hydrogels discovered here are synthetic, chemically defined and enable hESC expansion without the use of ROCK inhibitor.
This study demonstrates that simple synthetic hydrogels with three controllable parameters (adhesion peptides, degradability and stiffness) can outperform a natural biomaterial with over 1,500 unique proteins. The versatility of synthetic hydrogels, as demonstrated in the vascular biology and stem cell biology applications here, can be applied to a number of emerging biomanufacturing applications. For example, the hydrogels can be used to construct increasingly complex multicellular tissue models for drug and toxicity screening. Examples include vascularized tissues46,72, hierarchically assembled organoid models73 and models of developing embryonic tissue74. In this context, the synthetic materials can offer improved reproducibility and superior screening performance. In addition, synthetic hydrogels can provide a reproducible, chemically defined, xeno-free environment for the production of stem cells and their derivatives. Examples include pluripotent stem cells (described here), mesenchymal stem cells and other emerging therapeutic candidates. In this context, synthetic hydrogels can control the phenotype of the manufactured cells and decrease cost. Taken together, we predict that the use of synthetic hydrogel-based culture systems will ultimately lead to a significant reduction in false positives and false negatives in drug and toxicity screening, improved performance of compounds taken to preliminary animal testing, and an improvement in the safety and efficacy of cell therapies.
Endothelial-cell culture and maintenance
The HUVECs were purchased from Lonza and cultured in growth medium consisting of medium 199 (M199; Mediatech) supplemented with EGM-2 Bulletkit (Lonza). The medium supplement contained 2% fetal bovine serum, as well as hydrocortisone, human basic fibroblast growth factor, VEGF, R3-IGF-1, ascorbic acid, heparin, fetal bovine serum, human epidermal growth factor and GA-1000. The growth medium was changed every other day and the cells were passaged every five to seven days. Cell passages were performed using 0.05% trypsin solution (Thermo Fisher Scientific) and detached cells were recovered in M199 supplemented with 10% cosmic calf serum (Thermo Fisher Scientific). All media were supplemented with 100 U ml–1 penicillin and 100 μg ml–1 Streptomycin (Thermo Fisher Scientific). The cells were maintained in a humidified 37 °C incubator with 5% CO2 and used in between 5 and 16 population doublings in all experiments.
iPSC-ECs were provided by Cellular Dynamics International and cultured in complete VascuLife medium (Lifeline Cell Technology) supplemented with 10% fetal bovine serum and iCell Endothelial Cells Medium Supplement (Cellular Dynamics International). Culture flasks were coated with 30 μg ml–1 human plasma fibronectin (Corning) for 30 min before use. The growth medium was changed every other day and passaged every three to four days. The cells were detached using 0.05% trypsin and recovered in basal medium supplemented with 10% cosmic calf serum. The cells were maintained in a humidified 37 °C incubator with 5% CO2 and used in between 5 and 16 population doublings in all experiments.
hESC culture and maintenance
WA09 H1 hESCs (WiCell Research Institute) at passage 34 were thawed in E8 medium (Life Technologies) containing 5 μM ROCK inhibitor Y-27632 (EMD Millipore). Tissue-culture polystyrene plates were coated using Matrigel at a density of 0.0087 μg cm−2. The hESCs were seeded onto the plates and cultured for 24 h, then maintained in E8 medium at 37 °C in a 5% CO2 atmosphere. During routine maintenance, the media were changed every day and colonies showing morphological differentiation were manually removed before media change.
For colony passaging, the cells were incubated in Versene (Life Technologies) for 2–3 min at 37 °C until the edges of the colonies were weakly detached. Weakly adherent colonies were collected and centrifuged at 200 × g for 5 min, and the colonies were seeded between a 1:10 to 1:6 split ratio onto fresh Matrigel-coated plates.
For single-cell passaging, the cells were incubated in TrypLE (Life Technologies) for 3–4 min at 37 °C until the majority of the colonies were lifted. To dilute the TrypLE, 3 ml of E8 medium with 5 μM Y-27632 per 1 ml of TrypLE were added to TrypLE-treated cells. Afterwards, the cells were collected, centrifuged at 200 × g for 5 min, resuspended in E8 and 5 μM Y-27632 and mixed to singularize. Cells were seeded at 5,000 cells cm−2 onto fresh Matrigel-coated plates.
Labeling cells with CellTracker Red
One day before seeding the HUVECs/iPSC-ECs onto the hydrogel spots, the cells were stained with CellTracker Red to aid in the automated tubulogenesis quantification. Briefly, the HUVECs were rinsed in basal M199 for 5 min and stained with 1.3 μM CellTracker Red (Invitrogen) in M199 for 45 min. Afterwards, the cells were rinsed again in basal M199 for 5 min before incubation in growth medium overnight to allow the cells to sufficiently recover.
PEG functionalization with norbornene
Modification of PEG–OH molecules with terminal norbornene groups was performed using methods similar to those used in previous studies72,75. Briefly, PEG–OH (20 kDa molecular weight; eight-arm; tripentaerythritol core; JenKem Technology), dimethylaminopyridine and pyridine (Sigma Aldrich) were dissolved in anhydrous dichloromethane (Fisher Scientific). In a separate reaction vessel, N,N′-dicyclohexylcarbodiimide (Thermo Scientific) and norbornene carboxylic acid (Sigma Aldrich) were dissolved in anhydrous dichloromethane and reacted for 30 min to activate the norbornene. Norbornene carboxylic acid was covalently coupled to the PEG–OH through the carboxyl group by combining the PEG solution and norbornene solutions and stirring the reaction mixture overnight under anhydrous conditions. Urea byproduct was removed from the reaction mixture using a glass fritted funnel and the filtrate was precipitated in cold diethyl ether (Thermo Fisher Scientific) to extract the norbornene-functionalized PEG (PEGNB). The precipitated PEGNB was collected and dried overnight in a ceramic fritted filter. To remove impurities, the PEGNB was dissolved in chloroform (Sigma Aldrich), precipitated in diethyl ether and dried a second time in a Büchner funnel. To remove excess norbornene carboxylic acid, PEGNB was dissolved in deionized water, dialyzed in deionized water for one week and filtered through a Millex 0.45 μm pore-size polyvinyl difluoride syringe filter (Millipore). The aqueous PEGNB solution was frozen using liquid nitrogen and lyophilized. Functionalization of PEG was quantified using proton nuclear magnetic resonance spectroscopy to detect protons of the norbornene-associated alkene groups located at 6.8–7.2 ppm. The functionalization efficiency for norbornene coupling to the PEG–OH arms was above 90% for all PEGNB used in these experiments.
Preconjugation of adhesion peptides to PEG
Lyophilized PEGNB was dissolved in deionized water at a concentration of 0.5 mM and combined with 0.05% w/v Irgacure 2959 photoinitiator (I2959; Ciba Specialty Chemicals), as well as a 2× molar excess of either head-to-tail cyclized Arg–Gly–Asp–[d-Phe]–Cys (cyclic RGD; Genscript) adhesion peptide, 2× molar excess of linear H–Cys–Arg–Gly–Asp–Ser–NH2 (linear RGD; Genscript), 2× molar excess of non-functional H–Cys–Arg–Asp–Gly–Ser–NH2 scrambled adhesion peptide (CRDGS; Genscript), 3× molar excess of H–Cys–Glu–[d-Phe]–[d-Ala]–[d-Tyr]–[d-Leu]–Iso–Asp–Phe–Asn–Trp–Glu–Tyr–Pro–Ala–Ser–Lys–NH2 (VBP) or 3× molar excess of the non-functional scrambled VBP peptide H–Cys–Asp–[d-Ala]–Pro–Tyr–Asn–[d-Phe]–Glu–Phe–Ala–Trp–Lys–[d-Tyr]–Iso–Ser–[d-Leu]–Glu–NH2. The mixtures were reacted under 365 nm ultraviolet light for 5 min at a dose rate of 4.5 mW cm−2 to covalently attach the peptides to the norbornene groups via the thiol-ene reaction76. To remove buffer salts and unreacted peptides from the decorated PEGNB, the reaction mixtures were dialyzed in deionized water for two days. The dialyzed solutions were frozen in liquid nitrogen and lyophilized. The coupling efficiency of peptides to the PEGNB was quantified using proton nuclear magnetic resonance spectroscopy to detect disappearances of alkene protons at 6.8–7.2 ppm caused by covalent bonding of the peptides to the norbornene group (Supplementary Fig. 14).
Preparation of patterned gold slides with hydrophobic and hydrophilic regions
Gold-coated glass slides (EMF) were sonicated in 100% ethanol for 5 min and immersed in a 0.1 mM FlouroSAM solution (HS–C11–O–C2–(CF2)5–CF3; Pro Chimia) prepared in 100% ethanol for 2 h under protection from light at room temperature. This created a hydrophobic region on the surface of the gold. A polydimethylsiloxane mask with the pattern of choice was aligned with the gold slide and adhered to the surface. The exposed hydrophobic regions of the mask were etched by surface plasma treatment using a Diener Plasma Treatment Chamber (Diener Electronic) at 40 sccm and 50 W for approximately 1 min. After etching, the polydimethylsiloxane mask was removed and the slides were rinsed in 100% ethanol. The etched gold slide was placed into a 0.25 mM solution of [HS–C11–(O–CH2–CH2)3–OH] (EG3–OH) in 100% ethanol for 2 h at room temperature to create hydrophilic regions on the surface of the gold slide.
Silanization of glass slides for the screening of substrate conditions
Glass slides were sonicated for 45 min in 100% acetone to remove any surface impurities. The slides were rinsed three times in 100% ethanol to remove excess acetone from the surface. The cleaned slides were then surface plasma treated on both sides using the Diener Plasma Treatment Chamber for 5 min at 40 sccm and 50 W. The activated slides were transferred to 2.5% 3-mercatopropyl trimethoxylsilane (Sigma Aldrich) in toluene overnight. After retrieval, samples were cleaned by subsequent rinses with toluene at a 1:1 ratio of toluene to ethanol and three rinses of 100% ethanol, respectively. The slides were cured in a nitrogen-purged pressure chamber at 100 °C for 1 h. After curing, the silanized glass slides were placed in an airtight container and protected from light until use.
Preparation of PEG solutions for array formation
The hydrogels used for assessing endothelial-cell-network formation consisted of PEGNB molecules, PEG molecules preconjugated to RGD adhesion peptides and VBP, MMP-degradable H–Lys–Cys–Gly–Gly–Pro–Gln–Gly–Ile–Trp–Gly–Gln–Gly–Cys–Lys–NH2 crosslinking peptide (Genscript) and 0.05% w/v I2959 photoinitiator dissolved in phosphate buffered saline (1× PBS). The PEGNB, adhesion peptide and VBP concentrations were adjusted to varying levels to achieve different levels of stiffness, cell adhesion and VEGF-binding capabilities in the hydrogels. Before inclusion in precursor solutions, the concentration of the crosslinking peptide was verified using the Ellman’s Assay (Thermo Fisher). To vary endothelial-cell adhesion to the hydrogels, precoupled PEGNB–linear RGD and PEGNB–cyclic RGD molecules were added to the solutions to achieve the desired adhesion peptide concentrations between 0 and 4 mM. PEGNB–CRDGS molecules were added along with the functional adhesion molecules to provide a total of 4 mM pendant adhesion peptide in every solution. To vary VEGF binding to the hydrogels, precoupled-PEGNB–VBP molecules were added to the solutions to achieve desired peptide concentrations of 0.3 mM VBP. To vary the modulus of the hydrogels, PEGNB was added at 2.0, 2.5 or 3.5 mM concentrations and crosslinking molecules were added to the solution to achieve crosslinking of 50% of the total norbornene functional groups present in the solution. Once completed, all the hydrogel solutions were immediately stored at −80 °C, thawed immediately before use and subjected to only one freeze–thaw cycle before use.
The hydrogels used for assessing hESC pluripotency consisted of PEGNB molecules, PEG molecules preconjugated to cyclic RGD adhesion peptides, and 3.4 kDa dithiolated PEG crosslinking molecule (Laysan Bio) and I2959 photoinitiator dissolved in 1× PBS. The PEGNB and adhesion peptide concentrations were adjusted to varying levels to achieve different levels of stiffness and cell adhesion capabilities in the hydrogels. To vary hESC adhesion to the hydrogels, precoupled PEGNB–RGDFC molecules were added to the solutions to achieve the desired adhesion peptide concentrations between 0 and 4 mM. PEGNB–CRDGS molecules were added along with the functional adhesion molecules to provide a total of 4 mM pendant adhesion peptide in every solution. To vary the modulus of the hydrogels, PEGNB was added at 2.0, 2.5 or 4 mM concentrations and crosslinking molecules were added to the solution to achieve crosslinking of 50 or 75% of the total norbornene functional groups present in the solution. Once completed, all the hydrogel solutions were immediately stored at −80 °C, thawed immediately before use and subjected to only one freeze–thaw cycle before use.
Mechanical properties of PEG hydrogels
Shear modulus was measured in bulk samples of hydrogel spot formulations. To measure shear modulus, 72 μl measurements of hydrogel solution, each containing 0.125 mM RGDFC, were pipetted into Teflon wells of 8.0 mm diameter and 1.2 mm depth and cured for 8 s using 365 nm ultraviolet light at a dose rate of 90 mW cm−2. The resulting hydrogels were swollen in 1× PBS for 24 h and cut, if necessary, to a final diameter of 8 mm using a hole punch. The samples were tested using an Ares-LS2 rheometer (TA Instruments). A 20 g force was applied to the samples via parallel plate crossheads and a strain sweep test at a fixed frequency of 1Hz was performed from 0.1 to 20% strain. If the sample was not robust enough to withstand a 20 g force, the gap between the parallel plates of the rheometer was set to a distance of 1.0 mm instead. The complex shear modulus of each sample was the average of measurements taken at 10 Hz and 2 to 10% strain.
Preparation of thin hydrogel arrays for identifying hydrogels that promote endothelial-network formation
To prepare the silanized glass slides for formation of the hydrogel arrays, the samples were treated in 10 mM dithiothreitol (Sigma) in 1× PBS at 37 °C for 1 h to increase the number of free thiols on the surfaces of the slides. After incubation, the slides were sequentially rinsed in 1× PBS and 100% ethanol and dried with nitrogen gas. The patterned gold slides were rinsed with ethanol and dried using nitrogen gas. A 120-μm-thick polydimethylsiloxane spacer was applied to the surfaces of the gold slides to control the height of the hydrogels. Then, 0.8 μl of the prepared PEG solution was pipetted onto the hydrophilic spots of each glass slide in a humidity chamber75,77. A silanized glass slide was slowly placed onto the surface of each gold slide and transferred under an ultraviolet lamp. The hydrogel spots were exposed to 365 nm ultraviolet light at 4.5 mW cm−2 for 8 min. Following polymerization, the glass slides were removed from the underlying gold slides. This resulted in a patterned hydrogel arrays on the surfaces of the glass slides. Samples were stored in 1× PBS overnight at 4 °C before sterilization and seeding (Supplementary Video 4).
Assembling and seeding of hydrogel arrays with endothelial cells
On the day of culture, the arrays were assembled within three-chamber ProPlate Isolator assemblies (Grace Bio-Labs). The arrays were allowed to warm to room temperature, removed from the 1× PBS rinse and subsequently sterilized by immersion in 70% ethanol for 30 min, followed by two rinses in 1× PBS. Residual PBS was carefully aspirated from the regions surrounding the hydrogel spots to guarantee adherence to the Grace bio isolators. Using a sterile technique, the hydrogel arrays were subsequently assembled within the Grace bio isolator system and the individual wells were bathed in basal M199 until use. The cells stained with CellTracker Red were removed from the incubator and subsequently rinsed with 1× PBS. The cells were passaged by incubation in 0.05% trypsin (Hyclone) for 5 min, quenching of the enzyme through the addition of basal M199 with 10% cosmic calf serum, and subsequently counted and resuspended to give a cell count of approximately 85,000 cells cm−2. Residual medium was removed from the assembled arrays and replaced with the resuspended cell solution. When seeding the HUVECs, the cells were resuspended in M199 containing either 0, 5 or 10 ng ml–1 of VEGF and Endothelial Cell Growth Medium 2 Bulletkit (EGM2; without VEGF) as per Fig. 1d. When seeding the iPSC-ECs, the cells were resuspended in full VascuLife growth medium supplemented with 0, 5 or 10 ng ml–1 of additional VEGF. After seeding, the assembled constructs were transferred to a 37 °C incubator for 48 h. During this period, the hydrogel spots were imaged using a Nikon TI Eclipse inverted florescence microscope and individual spots were subsequently qualitatively scored on their ability to promote endothelial-network formation (Fig. 1d). Briefly, the spots were qualitatively assigned a score from 0 to 3, where 0 indicated no adhesion, 1 indicated low cell adhesion, 2 indicated monolayer formation and 3 indicated network formation. Each condition was tested using at least three spots per condition and a final qualitative score was assigned based on the behaviour observed in the majority of replicates.
Assembling and seeding of hydrogel arrays with embryonic stem cells
hESCs cultured on hydrogel arrays were seeded as colonies (at a split ratio of 1:10) or single cells (5,000 cells cm−2) in either E8 media (with or without 5 μM Y-27632) or maintained E8 media (with or without 5 μM Y-27632). The seeding media were replaced with the designated maintenance media 24 h after seeding. Media were changed on a daily basis for four days before fluorescence imaging was performed.
Quantification of hESC behaviour on hydrogel arrays
The hydrogel arrays were incubated on an environmentally controlled stage on the Nikon TI Eclipse microscope to match the atmospheric conditions of a humidified 37 °C incubator and photographed at 24, 48, 72 and 96 h after cell seeding. At 96 h, the cells were fixed via 15 min incubation in formalin. Immunofluorescence staining for NANOG was conducted using rabbit monoclonal antibodies to NANOG at a dilution of 1:400 (Abcam; Cat# ab109250) and Alexa Fluor 488 goat anti-rabbit secondary antibodies at a dilution of 1:200 (Life Technologies; Cat# A11008). Immunofluorescence staining for OCT3/4 was conducted using mouse monoclonal antibodies to OCT3/4 at a dilution of 1:100 (Santa Cruz Biotechnology; Cat# SC-5279) and Alexa Fluor 594 goat anti-mouse secondary antibodies at a dilution of 1:200 (Life Technologies; Cat# A11005). Immunofluorescence staining for SOX-2 was conducted using mouse monoclonal antibodies to SOX-2 at a dilution of 1:200 (Abcam; Cat# ab75485) and Alexa Fluor 594 goat anti-mouse secondary antibodies at a dilution of 1:200 (Life Technologies; Cat# A11005). Nuclei were stained using a 1:5,000 dilution of 4′,6-diamidino-2-phenylindole (DAPI; MP Biomedicals; Cat# 157574). The cell number was determined by the thresholding area of nuclei stained with DAPI, NANOG, OCT3/4 and SOX-2 using Nikon Elements Advanced Research version 4.13.00 (Build 914) LO 64 bit software (https://www.nikoninstruments.com/Products/Software/NIS-Elements-Advanced-Research) Percent marker expression was determined using automated measurements on the stained cells that were normalized against DAPI-stained cells.
Adapting network-forming hydrogels for use in 96-well plates
To ensure substrate stability and repeatable network area measurements in 96-well angiogenesis plates (ibidi), the photoinitiator concentration, solution volume and cell seeding density were optimized. For all hydrogel formation and cell seeding operations, screening arrays were constructed as follows: the 96-well angiogenesis plates were coated using 150–300 kDa poly(l-lysine) (PLL; Sigma Aldrich). A 0.01% v/v solution of PLL in deionized water was pipetted into the wells at a volume of 8 μl to evenly coat the bottoms of the wells. After 5 min of incubation at room temperature, the solutions were aspirated from the wells. Each well was then washed with deionized water three times before drying. The hydrogel solutions identified as enabling endothelial-network formation were pipetted into the wells, cured for 8 min under 365 nm, 4.5 mW cm–2 ultraviolet light and swollen in 70 μl 1× PBS overnight. For the HUVECs, the hydrogel solutions consisted of 2 mM PEG, 0.125 mM cyclic RGD and 4 mM crosslinking peptide. For the iPSC-ECs, the hydrogel solutions consisted of 2 mM PEG, 1 mM linear RGD and 4 mM crosslinking peptide. Afterwards, the PBS was aspirated and replaced with 35 μl of medium (network-forming medium was identified in the thin-hydrogel screening experiments) either by itself or containing vehicle or inhibitors at desired concentrations. For the HUVECs, the media consisted of M199 containing 5 ng ml–1 VEGF and EGM2 (without VEGF). For the iPSC-ECs, the media consisted of additional VEGF supplemented with 10 ng ml–1 of full VascuLife growth medium. Cell suspensions were added on top of the medium (35 μl of each). The cells were incubated in a humidified 37 °C incubator with 5% carbon dioxide for 24 h before the endothelial networks were photographed by epifluorescence and phase-contrast microscopy using a Nikon Eclipse microscope. After photography, the cells were fixed via 30 min incubation in formalin and prepared for confocal microscopy on a Nikon A1RS Confocal Microscope. Immunofluorescence staining for CD31 was conducted using mouse monoclonal antibodies to CD31 at a dilution of 1:200 (Dako; Cat# M082301-2) and Alexa Fluor 488 donkey anti-mouse secondary antibodies at a dilution of 1:200 (Life Technologies; Cat# A21202). Nuclei were stained using DAPI (MP Biomedicals; Cat# 157574) at a dilution of 1:5,000.
To enhance the substrate stability in the 96-well plates, the photoinitiator concentration was increased from the original 0.05% w/v concentration used in the thin-hydrogel arrays to 0.1, 0.2 and 0.4% w/v I2959. The swollen diameters of the hydrogels retrieved from the 96-well plates were measured to determine the hydrogel stabilities. Test hydrogels were cured at volumes of 8 μl per well using the aforementioned array assembly procedure. A 3.5-mm-diameter hole punch was used to retrieve hydrogel samples from the wells before the diameter of the samples was measured using a micrometer. Unstable hydrogels were evaluated as having the largest diameters after removal from the wells, indicating high swelling at equilibrium (Supplementary Fig. 6a).
To reduce the impact of meniscus formation on the imaging of the endothelial networks, 7, 8, 9 and 10 μl volumes of hydrogel were cured in the well plates using the aforementioned array assembly procedure. The hydrogels contained 0.2% w/v photoinitiator, as this had been found to be the optimal concentration in the swelling experiments. The HUVECs were stained with CellTracker Red and seeded onto the hydrogels at a density of 1.2 × 105 cells cm–2. Meniscus formation was evaluated by observing the in-focus and out-of-focus areas in a typical image of HUVEC network formation 24 h after seeding (Supplementary Fig. 6b).
To ensure the consistent formation of endothelial networks in the arrays, the HUVEC seeding density was explored at 1.2 × 105, 1.8 × 105 and 2.4 × 105 cells cm–2. The iPSC-EC seeding density was explored at 1.8 × 105 and 2.4 × 105 cells cm–2. The hydrogels were pipetted into the wells as 9 μl volumes, as this was determined to be optimal for meniscus reduction. Network formation was evaluated 24 h after seeding (Supplementary Fig. 6c).
Initial inhibitor screen for assessing optimized synthetic hydrogel assays in comparison with Matrigel
Synthetic hydrogel and Matrigel conditions were included in the screening arrays as follows: synthetic hydrogels were added to the 96-well plates in 9 μl volumes with HUVECs seeded at a density of 2.4 × 105 cells cm–2 and iPSC-ECs seeded at a density of 1.8 × 105 cells cm–2. Matrigel was added to the 96-well plates in 10 μl volumes and incubated in a humidified 37 °C incubator for 30 min. Afterwards, either the HUVECs or IPS-ECs were seeded at a density of 1.2 × 105 cells cm–2 using the aforementioned seeding procedure in 96-well plates. All the HUVEC experiments were conducted in M199 containing 5 ng ml–1 VEGF and EGM2 (without VEGF). All the iPSC-EC experiments were conducted in additional VEGF supplemented with 10 ng ml–1 of full VascuLife growth medium. Network formation was evaluated 24 h after seeding was performed in the 96-well plates. To conduct time-lapse microscopy of HUVEC network formation on the synthetic hydrogels or Matrigel, the HUVECs were seeded onto substrates as dictated for the initial inhibitor screens, treated with 0.2% dimethyl sulfoxide (DMSO) in medium, incubated on an environmentally controlled stage on the Nikon TI Eclipse microscope to match the atmospheric conditions of a humidified 37 °C incubator and photographed every 15 min using phase contrast microscopy.
The Z′ statistic (equation (1)) was used to assess the accuracy of the synthetic hydrogel and Matrigel screening system assays in distinguishing endothelial networks disrupted by the known angiogenesis inhibitor Sutent from non-inhibited networks. The equation accounts for differences in positive and negative controls, as well as s.d. across the assay40. PEG and Matrigel substrates were formed in separate 96-well plates. The HUVEC plates were divided into four 24-well corners with 0.1% DMSO controls occupying two opposite corners and 20 μM Sutent controls occupying the other two opposite corners. The iPSC-EC plates were divided into four 24-well corners with 0.2% DMSO controls occupying two opposite corners and 40 μM Sutent controls occupying the other two opposite corners. Endothelial networks were quantified using NIS-Elements Advanced Research version 3.20 (Build 677) 64 bit software, and a maximum size cutoff was applied to exclude the area of networks completely disrupted by Sutent.
The optimized PEG formulations with or without a VEGF binding component were directly compared with growth-factor-reduced Matrigel using an initial panel of five inhibitors: (1) Vatalanib, a clinically tested inhibitor to VEGF receptor activity78; (2) SU5416, a clinically approved inhibitor of VEGF receptor activity79; (3) sFlt-1, an antagonist of VEGF and placental growth factor80,81,82 ; (4) anti-VEGF, a human monoclonal antibody that binds soluble VEGF83; and (5) Sutent, a clinically approved inhibitor of VEGF receptor activity84,85. Endothelial cells were additionally treated on PEG and Matrigel substrates with inhibitors acting independent of VEGF signalling, including: (1) Prinomastat hydrochloride, an antagonist to the activity of MMPs 2, 3, 9, 13 and 14; and (2) Suramin hydrochloride, an inhibitor to epidermal growth factor, platelet-derived growth factor and transforming growth factor-β signalling16. Network formation was quantified and compared using the total endothelial-network area identified by the NIS Elements software, and a maximum size cutoff was applied to exclude the area of networks completely disrupted by Vatalanib or Sutent.
Screening of putative vascular inhibitors from the ToxCast library
To demonstrate the capability of the screening system to identify vascular inhibitors from a library of unknown chemical compounds, samples from the ToxCast library of chemicals were applied to the HUVECs similarly to the known vascular inhibitors screened on the 96-well plate. Of the 1,066 compounds in the ToxCast library, 38 chemicals (36 environmental chemicals and 2 reference compounds) were provided by the EPA to encompass a range of predicted activity levels, from ‘inactive’ to ‘strongly active’ inhibitory compounds based on a pVDC score from the ToxCast in vitro bioactivity profile1,46. The pVDC score had previously been generated from a filtered list of 23 assays and 309 chemicals (initially >500 in vitro assays and 1,066 chemicals) based on six biological assay targets related to embryonic blood vessel development in the ToxCast library using the Interactive Chemical Safety for Sustainability dashboard (http://actor.epa.gov/dashboard)41. The pVDC scores of the 38-compound test set are described elsewhere46,47. The compounds were presented as 53 samples, including replicates, the identities of which were not made known until after the data had been analysed. The compounds were assigned identification numbers (coded by the EPA and the National Center for Computational Toxicology in a database and only decoded after the data had been delivered and analysed) along with stock concentrations (25 mM or 100 mM, respectively). Although the samples were delivered as compounds in transparent 96-well plates and the colours of the stock solutions were visible to the experimenters, no other information was provided to reveal the identities of the unknown compounds. Additional details of the chemicals, quality assurance and control, and the assays performed can be found in ref. 86. The DMSO stock solutions from the library were diluted using tubulogenic medium at a dilution of 1:1,000 before they were added to the screening system. Endothelial-network inhibition was evaluated by quantifying the total area of endothelial networks treated with unknown compounds (a maximum size cutoff was applied to exclude the area of networks completely disrupted by Sutent) and comparing them with the mean total areas of non-inhibited control networks treated with vehicle only (0.2% DMSO). Networks with areas greater than 2 s.d. away from the mean were identified as being inhibited by a candidate compound. All the tests were performed in triplicate and compounds that resulted in inhibition for a majority of replicates were counted as inhibitors. These were given a binary output of either 1 for inhibited conditions or 0 for uninhibited conditions. Compounds identified as inhibitors in the majority of the triplicate plates were counted as inhibitors in the synthetic hydrogel and Matrigel systems.
All the data analysed were unpaired (that is, the samples were independent from each other). Before conducting multiple comparison tests, the Brown–Forsythe test was performed to determine the homogeneity of variance between the datasets. Normal distributions of 48 HUVEC and iPSC-EC network areas were determined using the Shapiro–Wilk normality test before inhibitor treatment. To compare multiple datasets, Tukey’s multiple comparisons test was used as a single-step multiple comparison procedure to find means significantly different from each other. To compare datasets with a DMSO or 1× PBS vehicle control, Dunnett’s test was used to find means significantly different from the control. All statistical tests were two-tailed (two-sided tests). All statistical analyses were performed using Graphpad Prism version 6.05 for Windows (http://www.graphpad.com/) software. P < 0.05 was considered significant. Variances between each group of data were represented by the s.d. Sample sizes to ensure adequate power were as follows: initial network formation screen, n = 3 sample replicates; Z′ tests, n = 48 sample replicates; known inhibitor panel, n = 3 sample replicates; DMSO and PBS controls, n = 8–12 sample replicates; blinded ToxCast screen, n = 3 repeated experiments with experimenters blinded to the chemical replicates during each experiment; hESC NANOG, OCT3/4 and SOX-2 expression and adhesion, n = 3 sample replicates (sample sizes were increased in conditions where cell viability was expected to be compromised, to n = 5 for colony seeding conditions where ROCK inhibitor was removed and n = 10 in single-cell seeding conditions where ROCK inhibitor was removed); mechanical, swelling, cell density and hydrogel volume optimization, n = 3 sample replicates; Ellman’s assays to determine free thiols in precursor solutions after freeze–thaw cycles, as well as before and after ultraviolet curing, n = 3 sample replicates; volumetric swelling assays, n = 3 sample replicates; and the detection of non-crosslinked PEG molecules after ultraviolet curing, n = 3 sample replicates. Samples were excluded from analysis if they were damaged during the testing procedure or if they were determined to be outliers through Grubbs’ outlier test.
The authors declare that all data supporting the findings of this study are available within the article and its Supplementary Information files. Source data for the figures in this study are available in figshare at http://dx.doi.org/10.6084/m9.figshare.c.3791386 (ref. 87).
How to cite this article: Nguyen, E. H. et al. Versatile synthetic alternatives to Matrigel for vascular toxicity screening and stem cell expansion. Nat. Biomed. Eng. 1, 0096 (2017).
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The authors acknowledge funding from the National Institutes of Health (NIH; R01HL093282-01A1, R21EB016381-01, 1UH2TR000506-01, T32HL007889, T32HL07936, R01EB10039, R24 EY022883, R01 EY026078, P30 EY016665, P30 CA 014520 and 5 P30 CA 014520-01), the Biotechnology Training Program (NIGMS5T32GM08349), the National Science Foundation (GE-0718123), the University of Wisconsin-Madison Graduate Engineering Research Scholars program, the Environmental Protection Agency (STAR grant no. 83573701), the Chemical Safety for Sustainability Research Program, the Office of Research and Development, the Virtual Tissue Models Project and the National Center for Computational Biology, the University of Wisconsin-Madison Molecular and Environmental Toxicity Center Training Program (NIH T32 ES007015), the Gates Millennium Scholars Program and the Retina Research Foundation, and an unrestricted departmental award from Research to Prevent Blindness. N.S. was a recipient of the Research to Prevent Blindness Stein Innovation Award. Mechanical testing data were obtained using the Ares LS2 rheometer at the University of Wisconsin-Madison Soft Materials Laboratory. This study made use of the National Magnetic Resonance Facility at Madison, which is supported by NIH grant P41GM103399 (NIGMS; old number: P41RR002301). Equipment was purchased with funds from the University of Wisconsin-Madison, the NIH (P41GM103399, S10RR02781, S10RR08438, S10RR023438, S10RR025062 and S10RR029220), the NSF (DMB-8415048, OIA-9977486 and BIR-9214394) and the United States Department of Agriculture. The authors acknowledge M. L. Dombroe for assistance with the HUVEC cultures and the laboratory of O. Mezu-Ndubuisi for providing mice for use in the aortic ring sprouting assays. The US Environmental Protection Agency (EPA), through its Office of Research and Development, funded and managed part of the research described here. The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the EPA.
Endothelial-network formation by HUVECs over the course of 24 hours after seeding onto PEG hydrogels.
Endothelial-network formation by HUVECs over the course of 24 hours after seeding onto Matrigel.
Endothelial-network formation by iPSC-ECs over the course of 24 hours after seeding onto PEG hydrogels.
Formation process of the thin hydrogel array.
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Nature Reviews Molecular Cell Biology (2017)