Tunable Synthetic Hydrogels to Study Angiogenic Sprouting

Angiogenic sprouting, the formation of new blood vessels from pre‐existing vasculature, is tightly regulated by the properties of the surrounding tissue microenvironment. Although the extracellular matrix has been shown to be a major regulator of this process, it is not clear how individual biochemical and mechanical properties influence endothelial cell sprouting. This information gap is largely due to the lack of suitable in vitro models that recapitulate angiogenic sprouting in a 3D environment with independent control over matrix properties. Here, we present protocols for the preparation of endothelial cell spheroid‐laden synthetic, dextran‐based hydrogels, which serve as a highly tunable 3D scaffold. The adjustment of the hydrogels’ adhesiveness, stiffness, and degradability is demonstrated in detail. Finally, we describe assays to elucidate how individual matrix properties regulate angiogenic sprouting, including their analysis by immunofluorescence staining and imaging. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.


INTRODUCTION
During angiogenic sprouting, endothelial cells are triggered to exit pre-existing blood vessels and form new vascular structures. This complex, multi-step process is regulated by many cues from the surrounding tissue microenvironment (Grainger & Putnam, 2013).
One critical regulator is the extracellular matrix (ECM), the three-dimensional network of proteins and polysaccharides, which not only provides structural support but also presents important biochemical and mechanical signals to residing cells. Here, matrix adhesiveness, stiffness, and degradability have emerged as important parameters regulating cell and tissue function (Madl & Heilshorn, 2018). However, how individual matrix properties affect angiogenic sprouting in 3D tissues remains largely unknown. Answering this question requires 3D hydrogel models, whose hydrated, porous architecture mimics the natural tissue microenvironment while offering full and independent control over individual matrix properties of interest.
In principle, hydrogels for in vitro models of angiogenic sprouting are based on either naturally occurring or synthetic materials. Natural hydrogels are composed of ECM proteins (such as type I collagen or fibrin) that self-assemble into hydrated fibrous 3D networks with micrometer-sized pores, and are characterized by inherent biocompatibility. Although such hydrogels are used in angiogenesis studies (Kim et al., 2013;Nguyen et al., 2013), it is difficult to tune one ECM parameter (e.g., stiffness) without concurrently affecting another one (e.g., adhesiveness) (Ingber, 1990). This limitation can be overcome through the use of synthetic hydrogels, which are based on an initially celland protein-inert polymer backbone that is subsequently functionalized with bioactive molecules of interest, thereby offering full control over ECM properties. Synthetic hydrogels are linear elastic (spring-like behavior) and mechanically isotropic (uniform stiffness in all directions) materials that are characterized by pores in the size range of tens to hundreds of nanometers (Trappmann & Chen, 2013;Trappmann et al., 2012). Additionally, their mechanical properties can be tuned to span the stiffness range of in vivo tissues (Discher et al., 2005;Engler et al., 2006;Yeung et al., 2005). Examples of commonly used fully synthetic or naturally occurring backbone polymers are poly(ethylene glycol), dextran, and hyaluronic acid.
In this article, we describe the preparation of a synthetic, dextran-based hydrogel with independent control over stiffness, adhesiveness, and matrix metalloproteinase (MMP)mediated degradability, and its use in angiogenic sprouting assays of embedded endothelial cell spheroids (ECSs). In this system, the invasion of endothelial cells into the surrounding hydrogel is initiated by a cocktail of pro-angiogenic factors, in a process that resembles in vivo angiogenic sprouting. The formation and extension of endothelial cell sprouts can be studied in real time, and matrix-dependent changes in sprout phenotypes can be directly correlated with a hydrogel property of interest.
In Basic Protocol 1, we report the synthesis and purification of methacrylated dextran (DexMA), the main constituent of our synthetic hydrogels. In Basic Protocol 2, we describe the preparation of ECSs as a model to examine angiogenic sprouting. ECSs are then embedded within DexMA hydrogels of varying stiffness in Basic Protocol 3, of different adhesive ligand densities in Basic Protocol 4, and of varying degradability in Basic Protocol 5. As a means to analyze endothelial cell sprouts within synthetic hydrogels, procedures for immunofluorescence staining and imaging of the 3D samples are outlined in Basic Protocol 6. Finally, Support Protocol 1 details the preparation of the pro-angiogenic cocktail used to drive endothelial cell sprouting.
NOTE: Although we have focused here on human umbilical cord vein endothelial cells (HUVECs) as a model cell type in this protocol, the techniques described are also directly applicable to other endothelial cell types, such as human microvascular cells (Liu et al., 2021).   The methacrylation of dextran involves the use of hazardous chemicals and must be performed in a chemical fume hood.

DexMA synthesis
1. Weigh 20 g dextran into a 250-ml round-bottom flask and add a stir bar.
2. Using a syringe, add 100 ml anhydrous DMSO while mixing on a magnetic hotplate. Immediately close the flask with a septum.

Pay attention to avoid skin punctures when retrieving DMSO from the glass bottle. Full dissolution of dextran in DMSO requires a few hours under stirring, and yields a very viscous solution.
3. After the dextran has completely dissolved, add 2 g DMAP.
4. After DMAP has dissolved, add 24.6 ml (1.5 equivalents relative to dextran repeating units) of GMA under vigorous stirring using a serological glass pipet with a rubber bulb. Ensure that the flask is well sealed with the septum, as the reaction is moisture sensitive.
As the addition of GMA forms a separate organic phase, it is recommended that the flask be manually shaken vigorously to quickly mix the two layers. The volume of GMA and the weight of dextran reported here are used to achieve a methacrylate/dextran repeating unit ratio of 0.7. For further information on how to obtain other degrees of functionalization (in molar equivalents relative to dextran repeating units), refer to Trappmann et al. (2017). 5. Place the round-bottom flask into an oil bath and heat to 45°C, using a temperature sensor for most precise heating. Stir the mixture vigorously for 24 hr and cover the round-bottom flask with tin foil to minimize exposure of the solution to light ( Fig. 2A).
The methacrylate group of GMA can readily react in the presence of light, and the flask should therefore be kept in the dark for the entire reaction time.
Over time, the solution will develop a darker color, turning dark brown after 24 hr ( Fig. 2A).

Current Protocols
DexMA purification and lyophilization 6. Cool down the solution to room temperature and precipitate DexMA by slowly pouring the reaction mixture into a beaker containing 1 L of cold (4°C) IPA under vigorous stirring.
During this step, a pale orange form (Fig. 2B). Make sure to cover the beaker containing the IPA solution with Parafilm during cooling to prevent moisture absorption. A high content of dissolved water in the IPA may affect the precipitation, leading to a loss in yield.
7. Transfer the precipitate containing solution to 50-ml conical centrifuge tubes and centrifuge 2 min at 1600 rcf, 4°C.
We recommend distributing the solution containing the precipitate across a maximum of 10 conical centrifuge tubes.
8. Discard the supernatant and repeat centrifugation of the pellets for 2 min at 1600 rcf, 4°C, to expel most of the solvent.
9. Resuspend the pellets in Milli-Q water.
If 10 conical centrifuge tubes were used for the precipitate retrieval, we recommend using ∼20 ml Milli-Q water per tube. 14. Lyophilize the frozen solution for ∼3 days to completely dry the polymer.
DexMA can be stored at −20°C for a few years.
16. Analyze the degree of methacrylate functionalization of the obtained product by 1 H-NMR spectroscopy using deuterium oxide as solvent.
To determine the degree of DexMA functionalization, calculate the average of the double bond proton integrals (6.2 and 5.7 ppm) and divide by the integral of the glucopyranosyl ring's anomeric protons (4.92 ppm), assuming a 4% ratio of α-1,3 linkages. For further details on the 1 H-NMR spectrum of DexMA, refer to Trappmann et al. (2017) and van Dijk-Wolthuis et al. (1995). This protocol should yield a degree of functionalization of ∼70% (∼0.7 methacrylate residues per dextran repeat unit). We recommend acquiring a 1 H-NMR spectrum of the product for each batch of DexMA to confirm the success of the functionalization.

GENERATION OF ENDOTHELIAL CELL SPHEROIDS IN MICROWELLS
The basis for all in vitro models of angiogenic sprouting are endothelial cells, which in vivo line the inner wall of blood vessels and are triggered to migrate into the surrounding matrix by chemokine gradients (Heiss et al., 2015). More specifically, HUVECs have evolved as a major primary model cell type as they can be easily obtained in large numbers (Jiménez et al., 2013). Historically, HUVECs were generally cultured on surfaces; however, this configuration fails to recapitulate the natural sprouting phenotype in a 3D tissue microenvironment and frequently results in the loss of their differentiated phenotype (Fina et al., 1990). The maintenance of a more natural phenotype, in particular, can be supported by a more physiological 3D culture setup, e.g., in the form of spheroids (Heiss et al., 2015).
Over the years, many different methods have been developed to generate ECSs reproducibly and efficiently. All techniques drive cells to self-aggregate by preventing their adhesion to an underlying culture substrate (Fang & Eglen, 2017). One common approach is the culture of endothelial cell suspensions as hanging drops, leading to cell aggregation and spheroid formation (Foty, 2011). Although this method does not require special equipment and is therefore easily accessible to many labs, it often results in a comparably low spheroid yield and a low degree of homogeneity. Instead, low-adhesion wells with U-or V-shaped geometries can be optimized for high spheroid shape reproducibility, yet here, again, the number of generated spheroids is generally low. To improve yield, spheroids can be generated in low-adhesion microwell plates. Here, a standardized array of microwells (e.g., 400 μm × 400 μm in size) permits the formation of many, highly uniform spheroids from cell suspensions.
This protocol aims to generate spheroids of HUVECs in a 24-well low-adhesion AggreWell TM microwell plate. Before the cell suspension is added, an anti-adherence rinsing solution must be applied to the well to prevent cell adhesion to the microwells and to initiate spheroid formation (Fang & Eglen, 2017). The input cell density can be adjusted to control the individual sizes of each of the ∼1200 ECSs that can be yielded per well. The cell density applied in this protocol will result in spheroids of ∼140 μm in diameter after an incubation period of 24 hr (Fig. 3). At this time, they can be harvested from the microwells and used for downstream applications. 2. Aspirate the anti-adherence solution and wash once with 2 ml PBS. Aspirate PBS and add 1 ml EGM-2 to the well.
4. Take up cells with 10 ml EGM-2 medium using a serological pipet and transfer the suspension to a conical centrifuge tube. Count cells in a counting chamber or equivalent. Prepare a cell suspension of 1.2 × 10 6 cells/ml or as desired (see AggreWell TM 400 datasheet) in a fresh conical centrifuge tube.

Generate spheroids
5. Add 1 ml of cell suspension to the EGM-2-containing well of the AggreWell TM 400 24-well plate prepared in step 2, to achieve a total volume of 2 ml. Mix gently by pipetting up and down to ensure an even distribution of cells throughout the well.
6. Centrifuge the plate for 3 min at 100 rcf, room temperature, to capture cells in the microwells along with the balance plate.
Pay attention to balancing the plate properly (well position, weight) to ensure an even distribution of the cells.
Observe the plate under a microscope to verify that cells are evenly distributed among the microwells (Fig. 3A). Each microwell should now contain about 1000 cells.
7. Incubate the plate at 37°C with 5% CO 2 and 95% humidity for 24 hr. Observe the cells under a microscope. Cells will assemble into spheroids after 24 hr (Fig. 3B).

ENDOTHELIAL CELL SPROUTING IN HYDROGELS OF TUNABLE STIFFNESS
Matrix stiffness is known to regulate endothelial cell migration patterns during angiogenic sprouting. In particular, we have shown that in both soft (∼0.8 kPa) and stiff (∼6 kPa) matrices, endothelial cells migrate as single cells, whereas cells acquire a collective migratory phenotype in matrices of intermediate stiffness (∼1.5 kPa) (Trappmann et al., 2017). Multicellular migration is required for subsequent vascular lumen formation, a critical step toward the formation of functional lab-grown blood vessels with in vivo-like properties (Liu et al., 2021; see Background Information for more details).
In Basic Protocol 3, we describe the preparation of DexMA hydrogels ( Fig. 4) with tunable stiffness, which are optimally designed to study the stiffness-dependent sprouting phenotype of ECSs. The protocol is divided into four sections: First, glass-coverslipsupported PDMS wells are prepared as holders for the spheroid-laden hydrogels; next, DexMA is functionalized with the integrin-binding peptide RGD to enable cell-matrix engagement; subsequently, the spheroid-laden precursor solution is crosslinked into MMP-cleavable hydrogels; and finally, cells are initiated to sprout by a pro-angiogenic cocktail (Fig. 5). DexMA functionalization with cell-adhesive peptides as well as hydrogel formation through coupling of MMP-cleavable peptide crosslinkers occur through the methacrylate groups introduced on the sugar backbone in Basic Protocol 1. Methacrylates undergo Michael-type addition with thiols present in cysteine residues of the peptides (mono-cysteine adhesive ligands, bis-cysteine crosslinkers; Fig. 4). Specifically, the presence of a base catalyzes the formation of nucleophilic thiolate anions, which react with methacrylates (Nair et al., 2014). DexMA hydrogel stiffness can be tuned by changing the concentration of MMP-cleavable peptides (Table 1; refer also to Trappmann et al., 2017 for stiffness values as a function of different crosslinker concentration). Changing hydrogel stiffness while keeping the content of DexMA polymer chains constant is a crucial design criterion of our system, because it ensures a comparable pore size and hence biomolecule diffusivity across the entire stiffness range (Trappmann et al., 2017). DexMA hydrogel stiffness can be measured through nanoindentation, a widely applied technique to probe the mechanical properties of materials, described in detail elsewhere (Ebenstein & Pruitt, 2006;Xu et al., 2022). An example of stiffness-dependent changes in endothelial cell migration mode is presented in Figure 6.    All recipes listed in Table 1 report the use of a bis-cysteine peptide crosslinker of native collagen degradability (NCD; sequence: CGPQGIAGQGCR, free N-and C-termini) and 6 mM linear RGD (sequence: CGRGDS, free N-and C-termini) as integrin-binding epitope. To prepare hydrogels of different degradability or adhesive epitope concentrations, refer to Basic Protocols 4 and 5, which describe the design criteria to adapt the recipes accordingly. NOTE: UV safety goggles need to be worn during UV sterilization of glass-coverslipsupported PDMS wells. All steps of hydrogel preparation need to be performed in a biological safety cabinet.

Preparation of glass-coverslip-supported PDMS wells
1. Follow the manufacturer's instructions and pour the desired volume of liquid PDMS pre-polymer solution into a plastic cup. Add the dedicated curing agent at a 1:10 ratio using a plastic pipet and mix thoroughly for several minutes.
As the two components are mixed, many bubbles will form.
Polydimethylsiloxane (PDMS) is an inert, nontoxic, and optically clear silicone-based elastomer (Raj & Chakraborty, 2020 3. To prepare PDMS wells, extract the solid PDMS layer from the dish with tweezers. Cut out circular pieces of PDMS using a 10-mm-diameter biopsy punch or, alternatively, 2 cm × 2 cm squares using a razor blade on a cutting mat.
4. Cut out holes of 5 mm diameter in each piece of PDMS. These cavities will function as hydrogel chambers.
5. Remove dust and particles from the PDMS pieces using sticky tape and attach each PDMS piece to a glass coverslip, ensuring conformal contact (24 × 24-mm square coverslip for square pieces or 15-mm circular coverslips for circular pieces). 6. Sterilize the glass-coverslip-supported PDMS wells using a UV light source (e.g., Omnicure® Series 1500 UV Spot Curing System, 10 mW/cm 2 for 60 s; intensity measured at 365 nm).

UV-sterilized glass-coverslip-supported PDMS wells can be stored indefinitely inside a tissue culture dish and used directly when required.
Linear RGD coupling to DexMA: First step of DexMA hydrogel preparation 7. Follow Table 1 to select the stiffness of interest.
8. Thaw aliquots of linear RGD and DexMA and equilibrate to room temperature.
9. Centrifuge the aliquots to spin down any liquid left on the walls of the vessels.
10. Add the required volume of DMEM, pH 7.0, to a sterile 0.5-ml reaction tube. 14. Dispense the required volume of DexMA solution into the reaction tube containing the mixture of DMEM and RGD and pipet up and down several times to wash the inner side of the tip. During the addition of NaOH solution, it is important to quickly homogenize the reaction mixture in order to avoid a locally high pH, which can lead to a local induction of the coupling reaction resulting in hydrogel inhomogeneity. The addition of NaOH solution will induce a shift of the reaction mixture color from yellow to pink once pH 8 is reached (Fig. 7A).
16. Allow the reaction to proceed for 30 min at room temperature.

Formation of ECS-laden hydrogels: Second step of DexMA hydrogel preparation
17. Transfer a glass-coverslip-supported PDMS well to a 10-cm tissue culture dish with an adhesive surface.
We recommend the use of a 10-cm tissue culture dish coated with cured PDMS (described in steps 1 and 2). The stickiness of the PDMS layer is required to keep the PDMS well in place.
18. Wet a Kimwipe with 2 ml antibiotic/antimycotic solution in PBS, fold it to obtain a long strip, and fit it inside the PDMS-coated dish by pressing it against the walls of the dish (Fig. 7B).
Kimwipes soaked with antibiotic/antimycotic maintain a moist environment within the dish, avoiding drying of DexMA hydrogel surface during polymerization; antibioticantimycotic is present to minimize risks of contamination.
19. In a biological safety cabinet, cut off the end of a 200-μl pipet tip using a razor blade. 27. Add the required volume of ECS suspension (from step 23) using a cut tip and pipet up and down gently to homogenize the solution.
28. Quickly cast the hydrogel precursor solution, ∼20 μl, within the glass-coverslipsupported PDMS well using a cut 200-μl tip, and flip the dish repeatedly to ensure an appropriate positioning of the spheroid until the hydrogel has formed (Fig. 7C). Let the hydrogel polymerize fully for 30 min outside the biological safety cabinet. Cover the outside of the dish with wet tissues to create a moisturized environment for polymerization.

ENDOTHELIAL CELL SPROUTING IN HYDROGELS OF TUNABLE ADHESIVENESS
In blood vessels, the endothelium is supported by a surrounding basement membrane consisting mainly of ECM proteins, such as laminin, type IV collagen, and heparan sulfate proteoglycans (HSPGs). Importantly, not only does this basement membrane serve as a support structure for endothelial cells, but its biochemical properties directly affect endothelial cell function, e.g., by regulating integrin activation through the presentation of adhesive ligands (Leclech et al., 2020). To study the regulatory role of individual adhesive ligands in vitro, synthetic hydrogels can be functionalized with peptides recapitulating the adhesive sites presented to endothelial cells in natural basement membranes. This approach offers an advantage over commonly used hydrogels of natural origin, such as type I collagen and fibrin, which do not provide control over the selection and concentration of adhesive ligands. In our standard DexMA hydrogels (Basic Protocol 3), the engagement of endothelial cells with their surrounding ECM is mediated through an RGD peptide (extended sequence: CGRGDS, free N-and C-termini). This peptide activates both α v β 3 and α 5 β 1 integrins (Kapp et al., 2017), which are known regulators of endothelial cell function during angiogenic sprouting in vivo (Brooks et al., 1994;Francis et al., 2002). By using DexMA hydrogels functionalized with different concentrations of RGD, our lab was able to elucidate the role of ECM adhesiveness in the regulation of multicellular sprouting.

Trapani, Weiß and Trappmann
In particular, endothelial cells were only able to sprout multicellularly through matrices functionalized with high concentrations of linear RGD, whereas they adopted a singlecell migratory phenotype at low peptide concentrations (Liu et al., 2021).
In Basic Protocol 4, we report the recipes for the preparation of DexMA hydrogels of a constant stiffness (∼1.5 kPa) (see Basic Protocol 3) and varying concentrations of RGD ligand, ranging from 0.15 to 6 mM (Table 2)-a range of concentrations shown to support HUVEC adhesion in 2D (Liu et al., 2021). These recipes will allow users to test the effect of matrix adhesiveness on ECS angiogenic sprouting independently from stiffness (Fig. 8). To keep chemical properties of hydrogels constant across the entire RGD concentration range, the adhesiveness is tuned by varying the ratio of RGD to RGE ligands, the latter not eliciting integrin activation (Takahashi et al., 2007).
The strategy described in this protocol can be applied to any other adhesive ligand of interest, such as selective ligands for α 5 β 1 integrins (Liu et al., 2021)   Humidified 37°C, 5% CO 2 cell culture incubator NOTE: UV safety goggles need to be worn during UV sterilization of glass-coverslipsupported PDMS wells. All steps of hydrogel preparation need to be performed in a biological safety cabinet.
1. Prepare glass-coverslip-supported PDMS wells as described in Basic Protocol 3, steps 1-6. Table 2 and select the linear RGD concentrations of interest.

Follow
3. Thaw aliquots of linear RGD, RGE, and DexMA and equilibrate them to room temperature.
4. Centrifuge the aliquots to spin down any drops left on the walls of the vessels.
5. Add the required volume of DMEM, pH 7.0 (see Table 2), to a sterile 0.5-ml reaction tube.

ENDOTHELIAL CELL SPROUTING IN HYDROGELS OF TUNABLE DEGRADABILITY
Synthetic hydrogels are nanoporous polymer networks that require active cleavage of crosslinker moieties to allow cell spreading and migration (West & Hubbell, 1999). Our DexMA hydrogels are crosslinked through peptide sequences that can be cleaved by specific sets of cell-secreted MMPs. The speed at which a given volume of covalently crosslinked hydrogel is cleaved by MMPs defines the degradability of the hydrogel. This degradability is affected by the concentration of the crosslinker as well as its susceptibility to MMP activity. Thus, in covalently crosslinked hydrogels, changes in stiffness through crosslinker concentration are always accompanied by differences in hydrogel degradability (Trappmann et al., 2017). In order to dissect the individual roles of these two parameters in endothelial cell sprouting, crosslinker peptides with different MMP susceptibility can be used to generate hydrogels of varying degradability but constant stiffness. Using this approach, our lab recently demonstrated that endothelial cells switch their migration pattern between single-cell and multicellular modes depending on the degradability of the matrix (Trappmann et al., 2017).
In Basic Protocol 5, we describe recipes for preparing DexMA hydrogels crosslinked with peptide sequences of different MMP susceptibility while keeping the stiffness (∼0.8 kPa) and adhesiveness (6 mM linear RGD) constant (Table 3). Specifically, a crosslinker sequence derived from the cleavage site of native collagen, called a "native collagen degradability" (NCD) sequence (CGPQGIAGQGCR, free N-and C-termini), introduced in Basic Protocols 3 and 4, is used as our standard sequence. Introduction of an amino acid mismatch to this sequence lowers its susceptibility to MMPs and therefore generates crosslinks of low degradability (LD, sequence: CGPQGPAGQGCR, free N-and Ctermini) (Nagase & Fields, 1996). The applicability of the resulting hydrogels is demonstrated by the degradability-dependent changes in endothelial cell migration modes in Figure 9.
The recipes reported in Basic Protocol 5 can be adapted to any other MMP-cleavable crosslinker processable by cell-secreted MMPs. A reference for MMP-cleavable peptide sequences is Turk et al., 2001. For further information on additional crosslinker peptides used in similar experimental conditions, refer to Liu et al., 2021.  and 631-1579) Omnicure® Series 1500 UV Spot Curing System (Excelitas Technologies Ltd) Water bath Set of single-channel pipets with respective tips Sterile 0.5-and 1.5-ml reaction tubes Kimwipes® (Kimtech Science, cat. no. 7552) Centrifuge Scale Spatulas Humidified 37°C, 5% CO 2 cell culture incubator NOTE: UV safety goggles need to be worn during UV sterilization of glass-coverslipsupported PDMS wells. All hydrogel preparation steps need to be performed in a biological safety cabinet. Table 3 and prepare hydrogels of desired degradability following the steps described in Basic Protocol 3. For hydrogels of low degradability, simply use the required volume of LD crosslinker solution instead of the NCD crosslinker.

IMAGING OF ENDOTHELIAL CELL SPHEROID-LADEN HYDROGELS
To gain insights into matrix-dependent signaling events, cultured ECSs within 3D DexMA hydrogel scaffolds can be assessed via immunofluorescence assays, including standard fixation methods, antibody staining, and fluorescence microscopy. Yet the surrounding nanoporous hydrogel hinders diffusion-based access to the cells. To achieve

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Current Protocols sufficient tissue preservation and antibody labeling, the application of all fixatives, antibodies, and fluorophores requires extended incubation times.
The imaging of embedded spheroids with a confocal fluorescence microscope requires the consideration of a set of additional parameters that may limit the acquisition results: Scattering of light by the cells and the hydrogel matrix, as well as mismatches in refractive indices between the hydrogel and the sample buffer (Riss & Trask, 2021). To reduce background noise from out-of-plane fluorescence, samples are best imaged with a confocal microscope with a uniform laser illumination. Due to the thickness of 3D samples, fast image acquisition with a spinning-disc microscope is advised. The objectives of choice must provide extensive working distances of up to several hundred micrometers to reach and image the spheroids embedded at distant z-layers within the hydrogel.
When assessing cells and spheroids in 3D, acquisition of structures close to the glass surface should be avoided. Cells can sense the stiffness of an underlying glass support surface up to a few tens of micrometers and thus must be excluded from the analysis in matrix-stiffness-related studies (Tusan et al., 2018). The imaging of ECSs close to the top of the hydrogel is advised, as these have better access to nutrients.

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Current Protocols 2. Wash three times with PBS, and add permeabilization solution (0.5% Triton-X in PBS) to permeabilize cells at room temperature for 1 hr.  10. Image the samples with a spinning-disc confocal microscope.

PREPARATION OF PRO-ANGIOGENIC COCKTAIL FOR ENDOTHELIAL CELL SPROUTING
Angiogenic sprouting in vivo is promoted by pro-angiogenic cues that activate endothelial cells. Three factors commonly associated with angiogenesis have been identified and confirmed to induce robust and reproducible sprouting in vitro: Vascular endothelial growth factor (VEGF), sphingosine-1-phosphate (S1P), and phorbol 12-myristate 13acetate (PMA) (Nguyen et al., 2013). VEGF and S1P both bind to their specific receptors on endothelial cells and activate downstream signaling cascades to promote cell migration and tube formation (Carmeliet & Jain, 2011;Takuwa, 2010). PMA passes through the plasma membrane and activates protein kinase C (PKC), which leads to the activation of important pro-angiogenic signaling pathways (e.g., ERK), resulting, for example, in elevated cell proliferation (Mebratu & Tesfaigzi, 2009;Verin et al., 2000;Wang et al., 1999).
To recapitulate the in vivo initiation of sprouting in the presented in vitro spheroid assay, a defined pro-angiogenic cocktail containing VEGF, S1P, and PMA at titrated concentrations is administered to the sample (Wang et al., 2020). A diffusion-based, chemoattractive gradient establishes within the synthetic hydrogel, triggering the endothelial cells to exit the spheroid body and invade the surrounding matrix.

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Current Protocols  PromoCell, cat. no. C-22211) and Supplement Pack (PromoCell, cat. no. C-39211) Biological safety cabinet 0.5-and 1.5-ml reaction tubes Set of single-channel pipets with respective tips NOTE: All steps of pro-angiogenic cocktail preparation need to be performed in a biological safety cabinet.
Prepare aliquots of pro-angiogenic cocktail components 1. Follow the manufacturer's instructions to reconstitute pro-angiogenic cocktail components as detailed in Table 4. Prepare stock solution aliquots in 0.5-ml reaction tubes. Store the tubes at −20°C; avoid freeze-thaw cycles. 3. Dilute stock solutions of S1P and PMA in warm EGM-2 as described in Table 4 to prepare intermediate solutions.

Preparation of pro-angiogenic cocktail
To increase pipetting accuracy, always pipet EGM-2 into a fresh tube first and add the precise volume of PMA or S1P in a second step. From our observations, cellular behavior is not altered by DMSO at dilutions >1:10,000. Table 4 to prepare the pro-angiogenic cocktail by combining stock solution of VEGF with intermediate solutions of PMA and S1P in warm EGM-2. For further details about the pro-angiogenic cocktail, refer to Nguyen et al. (2013).

DexMA aliquots (250 mg/ml in DMEM, pH 7.0)
Allow lyophilized DexMA to equilibrate from −20°C to room temperature. Weigh DexMA in a glass vial and dissolve in DMEM, pH 7.0 (Gibco, cat. no. 12800017), to a final concentration of 250 mg/ml. Dissolve at room temperature in the dark on a tube roller (a few hours will be required). Then, centrifuge the vial for 5 min at 1600 rcf, 4°C, to collect DexMA from the walls of the vessel. For sterile filtration, transfer the solution to Spin-X tube filters (0.22-μm-pore CA membrane; Costar, cat. no. 8160) in a biological safety cabinet and centrifuge 10 min at 20,000 rcf. Store aliquots in 0.5-ml reaction tubes under sterile conditions for up to 1 year at −80°C. Allow RGD powder (custom synthesized by Genscript at >95% purity) to equilibrate from −80°C to room temperature before opening the vial. Transfer RGD to a 2-ml reaction tube and dissolve in DMEM, pH 7.0 (Gibco, cat. no. 12800017)

Background Information
To examine the complex, multi-step process of angiogenic sprouting, a variety of experimental systems to observe and manipulate newly formed capillary vessels have arisen. Although in vivo models (e.g., mouse dorsal window chamber) capture the full complexity of blood vessel formation, it is very difficult to systematically study the role of individual signaling events or tissue properties of interest. Instead, in vitro cell culture models, such as the spheroid assay described here, have been developed to reduce this complexity by systematically mimicking and visualizing the most important steps of angiogenic sprouting in a well-controlled environment. These techniques have gradually evolved to include more and more relevant aspects, like a 3D scaffold with defined matrix properties and anatomical resemblance to the in vivo situation. Although the spheroid assay is a proven and reliable tool to recapitulate angiogenic sprouting, however, it does not capture other important steps of the in vivo process, such as directed sprouting towards a source of pro-angiogenic factors, continuous flow (Nguyen et al., 2013), and lumen formation. Biomimetic, microfluidic systems have recently emerged to manage these additional complexities (Nguyen et al., 2013). For example, Nguyen et al. developed a microfluidic platform consisting of a perfusable, cylindrical, endothelium-lined channel that is embedded within a surrounding type I collagen hydrogel. Application of a pro-angiogenic cocktail to a second, parallel channel generates a chemokine gradient

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Current Protocols through the hydrogel that triggers endothelial cells to sprout out into the matrix (Nguyen et al., 2013). Whereas historically such models were based on non-tunable, natural hydrogels, we have recently integrated synthetic DexMA hydrogels to study the impact of matrix properties on angiogenic sprouting (Trappmann et al., 2017). To further widen the scope of the methodology and also capture aspects of angiogenic sprouting that require longer culture periods, we replaced the hydrolytically labile methacrylates with more stable vinyl sulfone moieties. Using this modified hydrogel system, we were able to demonstrate the formation of perfusable, lumenized neovessels emanating from the parent channel (Liu et al., 2021). The vascular structures not only were functional in transporting fluid across the hydrogel, but also showed characteristic hallmarks of in vivo blood vessels, such as apical-basal polarity and deposition of basement-membrane-specific proteins, such as laminin and type IV collagen.

Critical Parameters
During the preparation of ECS-laden hydrogels, special attention needs to be paid to the pH of the precursor solutions upon addition of NaOH (in both the first and second steps), which can be monitored based on the color of an added pH indicator. Users should always aim for the same pH (8.0) to ensure a high degree of reproducibility. Indeed, differences in the pH of the precursor solutions may profoundly alter the kinetics of the Michaeltype addition reaction, ultimately affecting the final hydrogel stiffness and degree of adhesive ligand functionalization. Additionally, we recommend not exceeding pH 8 to avoid fast hydrolysis of the ester bonds present on the polymer chains, leading to the formation of more compliant hydrogels. Another important parameter to observe is the speed at which the NaOH solution is added to initiate the Michael addition. The solution needs to be mixed quickly to avoid a steep local increase in pH, which ultimately may lead to inhomogeneities within the hydrogel. Finally, attention needs to be paid to the dissolution of the bis-cysteine MMP-cleavable crosslinker peptides. The thiol groups of cysteine residues are prone to oxidation, leading to the formation of disulfide bonds that are no longer reactive towards methacrylates and thereby reducing the final stiffness of hydrogels. As oxidation of cysteine residues is likely to occur after dissolution of the MMP-cleavable crosslinker peptide powder, we highly recommend preparing the solution of this compound directly before it is required. Attention should be paid to the complete dissolution of the peptide. Moreover, appropriate storage and handling of the peptide in its powder form are also crucial: after thawing, always allow the powder to equilibrate to room temperature before opening the vial, and store the powder at −80°C again soon after use. Storage under inert gas (e.g., Ar or N 2 ) is also recommended. Table 5 lists common problems which may be encountered with our reported protocols. Each problem is listed together with its possible cause and solutions.

Understanding Results
Following this protocol, the researcher should be able to generate reproducible synthetic hydrogels with consistent mechanical properties. Mechanical testing of samples can be performed by nanoindentation, as shown by others (Ebenstein & Pruitt, 2006;Xu et al., 2022).
When performing the ECS sprouting assay, the researcher should observe mainly two migration phenotypes, depending on the hydrogel properties: single and collective cell migration. The expected experimental outcomes are summarized as follows: Effect of matrix stiffness at constant adhesiveness (6 mM RGD) and degradability (NCD crosslinker): • Single-cell migration is expected in a 0.8-kPa hydrogel; • Collective cell migration is expected in a 1.5-kPa hydrogel.
• Effect of matrix degradability at constant adhesiveness (6 mM RGD) and stiffness (0.8 kPa): • Single-cell migration is expected in a hydrogel with NCD crosslinker peptide; • Collective cell migration is expected in a hydrogel with LD crosslinker peptide.
• Effect of matrix adhesiveness at constant stiffness (1.5 kPa) and degradability (NCD crosslinker): • Single-cell migration is expected in a hydrogel containing 0.15 mM RGD; • Collective cell migration is expected in a hydrogel containing 6 mM RGD.
The migration mode can be evaluated by 3D confocal imaging of samples with fluorescently stained nuclei and F-actin using 3D analysis software (e.g., IMARIS x64, version 9.1.1, https:// imaris.oxinst.com). First, the total cell number can best be determined by  Repeat the preparation of the sample and increase the volume of 1 M NaOH. Subtract the additional volume from DMEM, pH 7.0, to keep a constant final volume of 90 μl.
Unusual morphology of angiogenic sprouts Cells that are not confluent Trypsinize a confluent dish of HUVECs with cobblestone pattern for the preparation of ECSs (no less than ∼4 million cells should be harvested from a 10-cm tissue culture dish).

Incorrect pro-angiogenic cocktail
Prepare fresh aliquots of pro-angiogenic cocktail components paying attention to the dilution factors and avoid freeze-thaw cycles.
Low fluorescence signal Too low antibody concentration Titrate the antibody concentration.

Insufficient incubation time with antibody staining solutions
Prolong incubation to allow for good diffusion of antibodies into the hydrogel sample.
High background signal Insufficient washing steps Replace washing buffer (PBS + 0.1% Tween 20) multiple times per day (at least three times).

Non-reproducible ECS-laden hydrogels
Inaccurate pipetting Pay careful attention when pipetting 1 M NaOH solutions and viscous DexMA.

Contamination
Inappropriate handling or sterilization of glass-coverslip-supported PDMS wells, tissue culture dishes, and/or solutions Sterilize solutions using Spin-X tube filters. Sterilize tissue culture dishes and glass-coverslip-supported PDMS wells with UV light.
an (automatic) detection of all nuclei within the cohort of migrated cells (IMARIS Spots assistant). Then, the number of cells (nuclei) in sprouts has to be manually counted and saved as subsets. An empirically determined threshold of six or more nuclei in a collective strand connected to the main body is advised as a measure for the interpretation of collectivity (Trappmann et al., 2017). If the connection of the sprout to the main body is lost, the respective cells are counted as single cells. The degree of collectivity is the ratio of the collectively migrating cells to the total number of migrating cells. If a high density of cells in close proximity complicates the judgement of collectivity, additional staining of the cell membrane through R18 membrane dye or cell junctions via antibodies against VE-cadherin or catenins may improve the evaluation.

Time Considerations
Synthesis and purification of DexMA (Basic Protocol 1) require 8 days in total: 1 day for the coupling reaction, 1 day for DexMA precipitation in IPA and dissolution in water, 3 days of dialysis against water, and 3 days of lyophilization.
ECS formation (Basic Protocol 2) takes ∼1 day: 1 hr for microwell coating and HUVEC trypsinization, counting, and seeding into microwells of a AggreWell TM 400 plate plus an additional 24 hr of culture in the cell culture incubator to allow cell aggregation.
Generation of ECS-laden hydrogel samples and angiogenic sprouting (Basic Protocols 3, 4, or 5) require ∼2.5 days total: 3 hr for hydrogel preparation, 4 hr of sample incubation with EGM-2 after DexMA gelation, and 48 hr of culture in presence of pro-angiogenic cocktail to induce sprouting. An additional 5 hr

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Current Protocols should be considered for the preparation of glass-coverslip-supported PDMS wells: ∼30 min for mixing of PDMS pre-polymer solution with curing agent, degassing, and distribution within tissue culture dishes, 4 hr for curing in the oven, and ∼30 min for fabrication of glasscoverslip-supported PDMS wells.
Staining of ECS-laden hydrogel (Basic Protocol 6) for visualization of nuclei and Factin requires ∼1 day: 1 hr for sample fixation, 1 hr for sample permeabilization, plus overnight incubation with Hoechst and phalloidin staining solution. If antibody staining needs to be performed, the staining procedure takes up to 6 days: 3 hr in total for fixation, permeabilization, and blocking (1 hr for each step), 1 day for primary antibody incubation followed by 2 days of washing, overnight incubation with secondary antibody followed by 1 day of washing, and finally overnight incubation with Hoechst and phalloidin solution.
Preparation of pro-angiogenic cocktail (Support Protocol 1) takes a few hours for the preparation of aliquots of VEGF, S1P, and PMA stock and intermediate solutions (∼1 hr for each component). The final dilution of VEGF, S1P, and PMA in EGM-2 for the pro-angiogenic cocktail preparation requires ∼10 min.