Name: standardized and scalable assay to study perfused 3d angiogenic sprouting of ipsc-derived endothelial cells in vitro
Uploaded: 2019-11-06T13:00:00.000+00:00
Duration: 10 min 47 s
Description: Watch this Scientific Journal Video about Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro at JoVE.com

This work is in part supported by a research grant from the &#8216;Meer Kennis met Minder Dieren&#8217; program (project number 114022501) of The Netherlands Organization for Health Research and Development (ZonMw) and the Dutch Heart Foundation CVON consortium grant RECONNECT.

1. Device Preparation
<ol>
	<li>Transfer the microfluidic 384-well plate to a sterile laminar-flow hood.</li>
	<li>Remove the lid and add 50 &#181;L of water or phosphate-buffered saline (PBS) to each of the 40 observation wells (Figure 1b, well B2) using a multichannel or repeating pipette. 
	NOTE: The protocol can be paused here. Leave the plate with the lid on in the sterile culture cabinet at room temperature (RT).</li>
</ol>
2. Prepare Gel and Coating
<ol>
	<li>Prepare 2.5 mL of 10 &#181;g/mL fibronectin (FN) coating solution. Dilute 25 &#181;L of 1 mg/mL fibronectin stock solution in 2.5 mL of Dulbecco&#8217;s PBS (dPBS, calcium and magnesium free). Place the solution in the water bath at 37 &#176;C till use.</li>
	<li>Prepare 100 &#181;L of collagen-1 solution. Add 10 &#181;L of HEPES (1 M) to 10 &#181;L of NaHCO3 (37 g/L) and mix by pipetting. Place the tube on ice and add 80 &#181;L of collagen-1 (5 mg/mL) to yield a neutralized collagen-1 concentration of 4 mg/mL. Use a pipette to mix carefully and avoid the formation of bubbles.</li>
	<li>Add 1.5 &#181;L of collagen-1 solution (4 mg/mL) to the gel inlet of each microfluidic unit (Figure 1b, well B1). Make sure the droplet of gel is placed in the middle of each well in order for the gel to enter the channel (see Figure 2a). 
	NOTE: Phaseguides prevent filling of the adjacent channels and enables gel patterning. Correct gel loading can be confirmed under a brightfield microscope by observing the meniscus formation through the &#8216;observation window&#8217; (well B2) or by flipping the plate upside down. If the gel did not completely fill the channel, an additional droplet of 1 &#181;L can be added.</li>
	<li>Place the microfluidic plate in an incubator (37 &#176;C, 5% CO2) for 10 min to polymerize collagen-1. 
	NOTE: Timing of the polymerization is crucial; due to the low volumes used in microfluidics, evaporation can already be observed after 15 min of incubation, which results in gel collapse or shrinkage.</li>
	<li>Take the plate out of the incubator and transfer to a sterile laminar-flow hood.</li>
	<li>Add 50 &#181;L of 10 &#181;g/mL FN coating solution to the outlet well of the top perfusion channel of every microfluidic unit (Figure 1b, well A3). Press the pipette tip against the side of the well for correct filling of the well without trapping air bubbles (see Figure 2b). The channel should fill, and the liquid should pin on the inlet (well A1) without filling the inlet well.</li>
	<li>Place the plate in the incubator (37 &#176;C, 5% CO2) for at least 2 days. 
	NOTE: The protocol can be paused here, as the collagen-1 gel together with the coating mixture is stable for at least 5 days in the incubator. If the coating mixture is refreshed, longer periods could be possible, but this has not been tested. Crucial is the level of FN-coating, as this prevents the dehydration of the collagen-1 gel.</li>
</ol>
3. Cell Seeding/Microvessel Culture
<ol>
	<li>Add 5 mL of fetal calf serum and 2.5 mL of pen/strep to 500 mL of basal endothelial cell culture medium and filter sterilize using a bottle top filter with 0.22 &#181;m pore size. This medium is now referred to as basal medium.</li>
	<li>Prepare vascular growth medium: Add 3 &#181;L of 50 &#181;g/mL vascular endothelial growth factor (VEGF) and 2 &#181;L of 20 &#181;g/mL basic fibroblast growth factors (bFGF) to 5 mL of basal medium.</li>
	<li>Thaw the frozen iPSC-ECs rapidly (&#60;1 min) in a 37 &#176;Cwater bath and transfer to a 15 mL tube and dilute in 10 mL of basal medium.</li>
	<li>Count the cells. 
	NOTE: A single vial contains 1 million cells in 0.5 mL with &#62;90% viability.</li>
	<li>Centrifuge the tube at 100 x g for 5 min. Aspirate supernatant without disturbing the cell pellet and resuspend in basal medium to yield a concentration of 2 x 107 cells/mL.</li>
	<li>Transfer the microfluidic 384-well plate from the incubator to a sterile laminar-flow hood.</li>
	<li>Aspirate FN-coating solution from the perfusion outlet well (A3) and replace with 25 &#181;L of basal medium in the outlet well (well A3).</li>
	<li>Add a 1 &#181;L droplet of cell suspension to every top perfusion inlet well (Figure 1b, well A1). The droplet should flatten in a few seconds (see Figure 3a,b for an illustration of this &#8216;passive pumping&#8217; method). 
	NOTE: Check under a microscope whether the seeding is homogeneous. If not, add another 1 &#181;L in the outlet and wait till the droplet flattens.</li>
	<li>Incubate the microfluidic well-plate for 1 h at 37 &#176;C, 5% CO2. After this, the cells should have adhered. If not, wait another 30 min.</li>
	<li>Remove the basal medium from the top perfusion outlet wells (Figure 1b, well A3). Add warm vessel culture medium in the top perfusion inlet and outlet (Figure 1b, wells A1 and A3). Place the plate on the rocker platform (set on 7&#176; angle, 8 min rocking interval) in the incubator (37 &#176;C, 5% CO2).</li>
	<li>Image the plate using a brightfield microscope with automated stage at day 1 and 2 post-seeding to confirm cell viability. After 2 days, a confluent monolayer should have formed against the collagen-1 scaffold. 
	NOTE: If the channels do not appear to be equally confluent, the microvessels can be cultured for an additional 24 h.</li>
</ol>
4. Study Angiogenic Sprouting Including Tip- and Stalk-cell Formation
<ol>
	<li>Prepare 4.5 mL of angiogenic sprouting medium by supplementing basal medium with 4.5 &#181;L of VEGF (50 &#181;g/mL stock), 4.5 &#181;L of phorbol 12-myristate-13-acetate (PMA) (2&#160;&#181;g/mL stock), and 2.25 &#181;L of sphingosine-1-phosphate (S1P) (1 mM stock).</li>
	<li>Prepare 8.5 mL of vessel growth medium (basal medium supplemented with 30 ng/mL VEGF and 20 ng/mL bFGF).</li>
	<li>Aspirate medium from the wells and add 50 &#181;L of fresh vessel culture medium in the top perfusion inlet and outlet wells and gel inlet and outlet wells (Figure 1b, wells A1, A3, B1 and B3).</li>
	<li>Add 50 &#181;L of angiogenic sprout mixture to each of the bottom perfusion channel inlet and outlet wells (Figure 1b, wells C1 and C3).</li>
	<li>Place the device back in the incubator (37 &#176;C, 5% CO2) on the rocker platform in order to form a gradient of angiogenic growth factors.</li>
	<li>Image 1 day and 2 days after addition of the angiogenic growth factors using a brightfield microscope with automated stage. 
	NOTE: Continue culturing the microvessels to study anastomosis (go to section 5) or fix and stain the microvessels to quantify sprouting length and morphology at day 2 and day 6 (go to section 6).</li>
</ol>
5. Study Anastomosis and Sprout Stabilization
<ol>
	<li>Image using a brightfield microscope with automated stage.</li>
	<li>Add 1 &#181;L of fluorescently labeled albumin (0.5 mg/mL) to the top perfusion inlet (Figure 1b, well A1) and mix using a 50 &#181;L pipette.</li>
	<li>Transfer the plate to a fluorescent microscope with automated stage and incubator set at 37 &#176;C. Set microscope at 10x objective and correct exposure settings (e.g., tetramethylrhodamine [TRITC]-channel, 20 ms exposure). Acquire time-lapse images every minute for 10 min.</li>
	<li>Remove the plate from the microscope and transfer the plate to a sterile laminar-flow hood.</li>
	<li>Remove all medium from the wells and replace both the vessel culture medium and angiogenic sprouting medium in the corresponding wells (see steps 4.3 and 4.4).</li>
	<li>Place the device back into the incubator (37 &#176;C, 5% CO2) to continue angiogenic sprouting.</li>
	<li>Repeat steps 5.1&#8722;5.6 to study the permeability at day 6.</li>
</ol>
6. Fixation, Staining, and Imaging
<ol>
	<li>Aspirate all culture media from all the wells. 
	NOTE: Residual medium or liquids in the microfluidic channels does not influence the fixation due to its low volume of 1&#8722;2 &#181;L.</li>
	<li>Add 25 &#181;L of 4% paraformaldehyde (PFA) in PBS to all the perfusion inlet (Figure 1b, A1 and C1) and outlet wells (Figure 1b, A3 and C3). Incubate for 10 min at RT. Place the device under a slight angle (&#177;5&#176;) to induce flow (e.g., by placing one side of the plate on a lid).</li>
	<li>Aspirate PFA from the wells. Wash all the perfusion inlets and outlets twice with 50 &#181;L of Hank&#8217;s balanced salt solution (HBSS). Aspirate HBSS from wells.</li>
	<li>Permeabilize at RT for 10 min by adding 50 &#181;L of 0.2% nonionic surfactant to all the perfusion inlets and outlets.</li>
	<li>Aspirate the nonionic surfactant from wells. Wash the perfusion channels twice by adding 50 &#181;L of HBSS to all perfusion inlet and outlet wells. Aspirate HBSS from wells.</li>
	<li>Stain the nuclei using Hoechst (1:2,000) and F-actin using phalloidin (1:200) in HBSS. Prepare 2.2 mL for 40 units, and add 25 &#181;L to each perfusion inlet and outlet well. Place the plate under a slight angle and incubate at RT for at least 30 min.</li>
	<li>Wash twice with 50 &#181;L of HBSS in all the perfusion inlets and outlets.</li>
	<li>Directly image using a fluorescent microscope with automated stage or store the plate protected from light at 4 &#176;C for later use.</li>
</ol>

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P. Vulto and T. Hankemeier are shareholders of Mimetas BV. V. Borgdorff and A. Reijerkerk are employees of Ncardia BV. V. van Duinen, W. Stam, V. V. Orlova and A.J. van Zonneveld have no disclosures.

This method describes the culture of 40 perfusable endothelial microvessels within a robust and scalable microfluidic cell culture platform. Compared to traditional 2D and 3D cell culture methods, this method shows how a physiological relevant cellular microenvironment that includes gradients and continuous perfusion can be combined with 3D cell culture with adequate throughput for screening purposes.
One of the major advantages over comparable microfluidic assays is that this method does not rely on pumps for perfusion but uses a rocker platform to induce continuous perfusion in all microfluidic units simultaneously. This ensures that the assay is robust and scalable: plates can be stacked on a rocker platform. Importantly, all microfluidic units remain individually addressable, which allows this method to be implemented within drug screening including the generation of a dose-response curve. Furthermore, without a pump, imaging and medium replacement is far simpler with less risk of (cross)-contamination.
Another advantage of this method is usage of a standardized, pre-manufactured platform, while comparable microfluidic cell culture platforms need to be fabricated by the end-users. This availability facilitates the adoption of this assay among other academic and pharmaceutical research groups, leading to standardization. Also, unlike microfluidic prototypes, the 384-well plate interface ensures compatibility with the current lab equipment (e.g., aspirators, plate handlers and multichannel pipettes), facilitating the integration within the current screening infrastructure.
There are several critical steps in performing this assay. The collagen-1 gel should completely fill the gel channel. During gel loading, this filling can be observed by inspecting the microfluidic channels either through the observation window (Figure 2a) or by flipping the plate upside down (as shown in Figure 1a). While filling, the collagen gel should remain in the center channel, without flowing into adjacent perfusion channels. We noticed that the quality of the collagen-1 gel is crucial for proper assay performance. Collagen-1 batches with too high viscosity will lead to incomplete filling of the gel channel. After 10 min of polymerization at 37 &#176;C, the gel should be homogenous and clear. If collagen-1 is not stored properly (e.g., due to fluctuating temperatures in the fridge), collagen will polymerize within the channels with clearly visible fiber formation. This can result in invasion of the ECs into the gel without addition of angiogenic factors, but without proper lumen development.
When the cells are seeded, the fibronectin coating solution is removed from the wells, leaving only the microfluidic channels filled with coating solution. Aspiration of the coating solution from microfluidic channels could cause gel disruption or gel aspiration. The cell suspension needs to replace/displace this coating solution. This works best when the cell suspension is seeded using the passive pumping method, as directly pipetting the cell suspension into the channels show less reproducible seeding densities.
As the microvessels form a stable monolayer against the gel, these small differences only result in different times needed for reaching confluency. Thus, the assay start point is determined by confluency rather than culture time. If necessary, the culturing time can be extended until a clear monolayer has been formed against the gel.
Within the wells, air bubbles can be trapped by incorrect filling of the wells (see Figure 2b). These air bubbles will restrict the flow of medium, even when the device is placed on a rocker platform, and result in collapse of the microvessel and improper gradient formation. Pressing the pipette tip against the side wall of the wells will increase the success of completely filling the well. If an air bubble is trapped within the wells, it can be removed by gently inserting a sterile pipette tip into the glass bottom. Air bubbles can also occur within the microfluidic channels. When medium has been removed from the wells, evaporation of medium is noticeable from the microfluidic channels after 30 min (due to the microliter volumes within the channels). Thus, medium changes are preferably performed as quick as possible. When medium is added in a channel with evaporated medium, air bubbles will be trapped within the microfluidic channels. These air bubbles within the microfluidic channels can be removed manually by placing a P20 pipette directly on either the inlet or outlet and forcing medium through the microfluidics from the opposite well. Successful removal of the air bubbles results in a small but noticeable decrease in volume in the other well. Table 1 lists common errors and how to troubleshoot them.
The lack of a pump is a limitation when continuous imaging is required, as the rocker platform limits the user to image at sequential time intervals. Furthermore, the perfusion of medium in this platform consists of bi-directional flow with low levels of shear stress, while vasculature in vivo is exposed to unidirectional flow with higher levels of shear stress. While we do not observe negative effects of the bi-directional flow with regards to the angiogenic sprouting, flow is an important biomechanical stimulus and preferably controlled. However, while there are commercially available pump setups, interfacing with the 384-well plate remains challenging and pump setups severely hamper the scalability of this assay.
The possibility to use iPSC-ECs to study angiogenic sprouting opens up new opportunities in disease modeling and drug research. In contrast to primary ECs, these cells can be generated in nearly limitless quantities with a stable genotype and by using genome editing techniques, cells can be generated that including gene knockouts and knock-ins. However, as the protocols to differentiate ECs from iPSC are relatively new, it is still unclear what leads to iPSC-ECs that best reflect primary ECs and which subtypes of EC are or can be generated. Also, there are still remaining questions regarding their relevancy. For example, do iPSC-ECs still exhibit the plasticity that is typical for endothelial cells? And to what degree do iPSC-derived cells respond to and interact with their cellular microenvironment? The standardized platform presented here could be used to answer some of these questions in order to further validate the usage of iPSC-derived ECs in vitro.
The most straight-forward future direction for this assay will be the integration of other cell types that play an important role during angiogenesis, such as pericytes and macrophages. This will facilitate the ability to study the role of macrophages during anastomosis between sprouts or the adherence of pericytes after capillary formation. Also, it is possible to culture various other cell types within or against an extracellular matrix (e.g., we have shown the culture of neurons and various epithelial structures such as proximal tubules and small intestines), which can be combined with the vascular beds generated using this method. Finally, it will be interesting to study angiogenic sprouting in synthetic hydrogels, as their defined composition further increases the standardization of the assay and allows tuning of stiffness and binding motives that affect cell-matrix interactions.
In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/59678">Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro</a>. The Protocol section was&#160;updated.
Step 2.6 of the Protocol was updated from:
Add 50 &#181;L of 10 &#181;g/mL FN coating solution to the outlet well of the top perfusion channel of every microfluidic unit (Figure 1b, well A3). Press the pipette tip against the side of the well for correct filling of the well without trapping air bubbles (see Figure 2b). The channel should fill, and the liquid should pin on the outlet (well C1) without filling the outlet well.
to:
Add 50 &#181;L of 10 &#181;g/mL FN coating solution to the outlet well of the top perfusion channel of every microfluidic unit (Figure 1b, well A3). Press the pipette tip against the side of the well for correct filling of the well without trapping air bubbles (see Figure 2b). The channel should fill, and the liquid should pin on the inlet (well A1) without filling the inlet well.
Step 3.7 of the Protocol was updated from:
Aspirate FN-coating solution from the perfusion inlet (well A1). Add 25 &#181;L of basal medium in the inlet wells (well A1).
to:
Aspirate FN-coating solution from the perfusion outlet well (A3) and replace with 25 &#181;L of basal medium in the outlet well (well A3).

An erratum was issued for:&#160;<a href="https://www.jove.com/t/59678">Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro</a>. The Protocol section was&#160;updated.

Erratum: Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

In vitro models play a fundamental role in the discovery and validation of new drug targets of vascular diseases. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance1, which hinders the translation of findings from in vitro to in vivo. Thus, to advance pre-clinical vascular drug research, improved in vitro models of vasculature are necessary that combine high-throughput screening with a physiologically relevant 3D cellular micro-environment.
Within the last decade, significant progress has been made to increase the physiological relevance of in vitro models of vasculature. Instead of culturing endothelial cells on flat surfaces such as tissue-culture plastics, endothelial cells can be embedded in 3D scaffolds, such as fibrin and collagen gels2. Within these matrices, the endothelial cells show a more physiologically relevant phenotype associated with matrix degradation and lumen formation. However, these models only demonstrate a subset of the many processes that occur during angiogenic sprouting as important cues from the cellular microenvironment are still lacking.
Microfluidic cell culture platforms are uniquely suited to further increase the physiological relevance of in vitro models of vasculature. For example, endothelial cells can be exposed to shear stress, which is an important biomechanical stimulus for vasculature. Also, the possibility to spatially control fluids within microfluidics allows the formation of biomolecular gradients3,4,5,6. Such gradients play an important role in vivo during the formation and patterning during angiogenesis. However, while microfluidic cell culture platforms have shown unparalleled physiological relevancy over traditional 2D and 3D cell culture methods, they often lack the necessary throughput, scalability and standardization that is required for drug screening7. Also, many of these platforms are not commercially available and require the end-users to microfabricate their devices prior to use8. This not only requires manufacturing apparatus and technical knowledge, but also limits the level of quality control and negatively affects reproducibility9.
To date, primary human endothelial cells remain the most widely used cell source to model angiogenesis in vitro10. However, primary human cells have a number of limitations that hinder their routine application in screening approaches. First, there is a limited possibility to scale up and expand primary cell-derived cultures. Thus, for large scale experiment, batches from different donors need to be used, which result in genomic differences and batch-to-batch variations. Second, after a few passages, primary endothelial cells generally lose relevant properties when cultured in vitro11,12.
Endothelial cells derived from human induced pluripotent stem cells (iPSC) are a promising alternative: they resemble primary cells, but with a more stable genotype that is also amenable to precise genome editing. Furthermore, iPSCs are able to self-renew and thus can be expanded in nearly unlimited quantities, which make iPSC-derived cells an attractive alternative to primary cells for usage within in vitro screening models13.
Here, we describe a method to culture endothelial cells as perfusable 3D microvessels in a standardized, high-throughput microfluidic cell culture platform. Perfusion is applied by placing the device on a rocker platform, which ensures robust operation and increases the scalability of the assay. As the microvessels are continuously perfused and exposed to a gradient of angiogenic factors, angiogenic sprouting is studied in a more physiological relevant cellular microenvironment. While the protocol is compatible with many different sources of (primary) endothelial cells14,15, we focused on using human iPSC-derived ECs in order to increase the standardization of this assay and to facilitate its integration within vascular drug research.

<table><tbody><tr><td>0.2-10 &amp;mu;L Electronic repeater pipette</td><td>Sigma</td><td>Z654566-1EA</td><td></td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>3-lane OrganoPlate</td><td>Mimetas</td><td>4003-400B</td><td></td></tr><tr><td>4% PFA in PBS</td><td>Alfa Aesar</td><td>J61899</td><td></td></tr><tr><td>Basal culture medium</td><td>NCardia</td><td></td><td></td></tr><tr><td>Collagen-1</td><td>Cultrex</td><td>3447-020-01</td><td></td></tr><tr><td>Combitips Advanced Biopur 2.5 mL</td><td>VWR</td><td>613-2071</td><td></td></tr><tr><td>dPBS</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Eppendorf Repeater M4 pieptte</td><td>VWR</td><td>613-2890</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F4759-1MG</td><td>1 mg/mL in milliQ water</td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025-050</td><td></td></tr><tr><td>Hoechst</td><td>Molecular Probes</td><td>H3569</td><td>10 mg/mL solution in water</td></tr><tr><td>iPSC-derived endothelial cells</td><td>NCardia</td><td></td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td>37 g/L in milliQ water</td></tr><tr><td>OrganoPlate Perfusion Rocker Mini</td><td>Mimetas</td><td></td><td>Rocker platform used to provide flow</td></tr><tr><td>Pen/Strep</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Phalloidin</td><td>Sigma</td><td>P1951</td><td>1 mg/mL in DMSO</td></tr><tr><td>PMA</td><td>Focus-Biomolecules</td><td>10-2165</td><td>2 &amp;mu;g/mL in PBS containing 0.1% DMSO</td></tr><tr><td>S1P</td><td>Ecehelon Biosciences</td><td>S-2000</td><td>1 mM in methanol containing 1% acetic acid</td></tr><tr><td>TRITC-albumin</td><td>Invitrogen</td><td>A34786</td><td></td></tr><tr><td>VEGF</td><td>Peprotech</td><td>450-32-10</td><td>50 &amp;mu;g/mL in water containing 0.1% BSA</td></tr></tbody></table>

standardized and scalable assay to study perfused 3d angiogenic sprouting of ipsc-derived endothelial cells in vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

The microfluidic 3D cell culture platform consists of 40 perfused microfluidic units (Figure 1a,b), which is used to study angiogenic sprouting of perfused microvessels against a patterned collagen-1 gel (Figure 1c). These microvessels are continuously perfused and exposed to a gradient of angiogenic growth factors (Figure 3a-d). The angiogenic sprouts can be either studied 2 days after gradient exposure or cultured for more than 5 days after gradient exposure to study anastomosis and sprout stabilization (see timeline, Figure 1d).
Seeding the iPSC-ECs using the passive pumping method should result in homogenous seeding densities (Figure 4a,b). Culture under continuous perfusion resulted in confluent microvessels in 2 days, with the cells completely lining the circumference of the microfluidic channel and the formation of a confluent monolayer against the patterned collagen-1 gel.
Exposure to a gradient of angiogenic factors resulted in directional angiogenic sprouting of the microvessels within the patterned collagen-1 gel (Figure 5a-g). Clear tip cell formation and invasion into the collagen-1 gel was visible 24 h after addition of the angiogenic gradient, while stalk cells including lumen formation were visible after 48 h (Figure 5a).
After fixation and staining, the capillary network can be visualized using phalloidin to stain F-actin and using Hoechst 33342 to stain the nucleus (Figure 5b,c). These sprouts can be quantified (e.g., shape and length14). Without addition of growth factors, no invasion into the collagen-1 gel should be observed (Figure 5d). Confocal imaging was used to determine the sprout diameter and to confirm lumen formation (Figure 5e-g).
The sprouts continue to grow towards the direction of the gradient and reach the opposite perfusion channel within 3&#8722;4 days after addition of angiogenic growth factors. This results in remodeling of the vascular network, with a clear reduction in the number of angiogenic sprouts (Figure 6a). Lumen formation was assessed by perfusion of the vascular network with fluorescently labeled macromolecules (e.g., albumin or dextrans). Perfusing the microvessels with 0.5 mg/mL labeled albumin before and after anastomosis revealed a clear difference in sprout permeability after 10 min (Figure 6b-e), which suggests that the capillaries stabilize and mature after anastomosis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59678/59678fig1.jpg" /> 
Figure 1: Microfluidic cell culture protocol for iPSC-derived microvessels. (a) The bottom of the microfluidic cell culture device is shown displaying the 40 microfluidic units that are integrated underneath the 384-well plate. Larger view displays one of the 40 microfluidic units. (b) Each microfluidic unit is positioned underneath 9 wells with 3 inlet wells and 3 outlet wells. The microfluidic channels are separated by ridges (&#8216;phaseguides&#8217;), which enable the patterning of hydrogels in the central channel (&#8216;gel channel&#8217;) while there is still contact with the adjacent channels (&#8216;perfusion channels&#8217;). (c) Method to culture a perfused microvessel within the microfluidic device, which is used to study gradient driven angiogenic sprouting through a patterned collagen-1 matrix. (d) Timeline for studying angiogenic sprouting and/or anastomosis. This figure has been modified from van Duinen et al.14. <a href="https://www.jove.com/files/ftp_upload/59678/59678fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59678/59678fig2.jpg" /> 
Figure 2: Loading procedures for gel and medium. (a) Examples of correct and incorrect gel deposition. Correct filing results in a patterned collagen-1 gel in the middle channel, which is subsequently polymerized. (b) Examples of correct and incorrect filling of the wells. Wells are filled in the order of 1&#8722;4 to prevent air-bubble trapping within the microfluidic channels. <a href="https://www.jove.com/files/ftp_upload/59678/59678fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59678/59678fig3.jpg" /> 
Figure 3: Continuous hydrostatic pressure driven flow and gradient stabilization. (a) Hydrostatic pressure differences between wells result in passive levelling and flow within the microfluidic channels. (b) Placing the device on a rocker platform set at 7&#176; and 8 min cycle time results in continuous, bi-directional perfusion within the microfluidic channels. (c) Gradients are formed by introducing two different concentrations within the wells, which are continuously refreshed by passive leveling. (d) Gradient visualization using fluorescein isothiocyanate (FITC)-dextran. Bi-directional flow stabilizes the gradient up till 3 days. Scale bar = 200 &#181;m. This figure has been modified from van Duinen et al.14. <a href="https://www.jove.com/files/ftp_upload/59678/59678fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59678/59678fig4.jpg" /> 
Figure 4: Passive pumping method for cell seeding. (a) Passive pumping is driven by pressure differences that are caused by differences in surface tension. This results in a flow from the droplet (high internal pressure) towards the reservoir (low internal pressure). (b) Time lapse of a droplet (gray outline) that is placed on top of the inlet (white outline) of the microfluidic channel (blue outline). Right after addition (Figure 4b, i), the droplet on top of the inlet shrinks (Figure 4b, ii: 1 s after addition; iv: 2 s after addition), which results in a flow towards the outlet. This continues until the droplet meniscus is pinned by the inlet (Figure 4b, iv). Scale bar = 400 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59678/59678fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59678/59678fig5.jpg" /> 
Figure 5: Robust 3D sprouting of iPSC-EC microvessels. (a) Sprouting of iPSC-EC over time. Microvessels were grown for 48 h (right) and then stimulated with an angiogenic cocktail containing 50 ng/mL VEGF, 500 nM S1P, and 2 ng/mL PMA. The first tip-cells that invade the collagen-1 scaffold (middle) are visible 24 h after exposure. The first lumens are visible (arrows) 48 h after exposure (right) while the tip-cells have migrated further in the direction of the gradient. (b) Array of 15 microvessels that were stimulated with VEGF, S1P and PMA for 2 days and stained for F-actin (yellow) and nuclei (blue). Scale bar = 200 &#181;m. (c) Stimulated microvessel (positive control). (d) Unstimulated microvessel (negative control). (e) The maximum projection of a single capillary within the gel. (f) Same as (g) but focused on the middle. Dotted line indicates the position of the orthogonal view in panel g. Scale bars (a-d: 200 &#181;m; e-g: 20 &#181;m). <a href="https://www.jove.com/files/ftp_upload/59678/59678fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59678/59678fig6.jpg" /> 
Figure 6: Visualization of angiogenic sprout permeability before and after anastomosis. (a) Anastomosis with basal channel triggers pruning and maturation of angiogenic sprouts. Closeup of capillary bed at 2, 4, 6 and 7 days after stimulation with angiogenic growth factors. (b) Angiogenic sprouts after 2 days after addition of angiogenic growth factors. Angiogenic sprouts are formed within gel, but are not yet connected to the bottom perfusion channel. (c) Perfusion of the microvessel with 0.5 mg/mL albumin-Alexa 555 solution. Fluorescent images obtained at 0 and 10 min. (d,e) Same as in panels b and c, but after 7 days of stimulation. Sprouts are connected to the other side and formed a confluent microvessel in the basal perfusion channel. Scale bars = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59678/59678fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Cause</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td rowspan="3">Collagen-1 does not enter or fill the channel completely</td>
			<td>Collagen-1 droplet is not placed on top of the inlet</td>
			<td>Carefully place the droplet on top of the inlet from the gel channel</td>
		</tr>
		<tr>
			<td>Volume of collagen-1 is too low</td>
			<td>Use 1.5 &#181;L of the gel to fill the channel completely</td>
		</tr>
		<tr>
			<td>Collagen-1 is too viscous</td>
			<td>Use another batch of collagen-1</td>
		</tr>
		<tr>
			<td>Collagen-1 flows into perfusion channels</td>
			<td>Collagen-1 is pipetted directly into the inlet of gel channel</td>
			<td>Carefully place the droplet on top of the inlet from the gel channel</td>
		</tr>
		<tr>
			<td rowspan="2">Collagen-1 is not clear/fiber formation</td>
			<td>Collagen-1 is not stored properly</td>
			<td>Store collagen-1 at 4 &#176;C, do not freeze</td>
		</tr>
		<tr>
			<td>NaHCO3 and HEPES are not mixed well before adding collagen-1</td>
			<td>Carefully mix the NaHCO3 and HEPES by pipetting before adding the collagen-1</td>
		</tr>
		<tr>
			<td rowspan="2">Droplet does not shrink using passive pumping method</td>
			<td>Droplet adheres to side of the well</td>
			<td>Aspirate droplet and add new droplet on top of the inlet</td>
		</tr>
		<tr>
			<td></td>
			<td>Make sure the outlet well is filled with at least 20 &#181;L of medium</td>
		</tr>
		<tr>
			<td rowspan="3">No sprouting is observed</td>
			<td>Growth factors are not added or aliquots are not stored properly</td>
			<td>Prepare fresh angiogenic sprouting medium</td>
		</tr>
		<tr>
			<td>Air bubble blocks perfusion/ gradient formation</td>
			<td>Remove air bubbles using a P20 or P200 pipette</td>
		</tr>
		<tr>
			<td>Volume differences between wells</td>
			<td>Volumes in all wells need to be equal in order to form a linear gradient</td>
		</tr>
		<tr>
			<td rowspan="2">Cells not viable</td>
			<td>Plate not placed on rocker platform/rocker platform turned off</td>
			<td>Make sure rocker platform is on and has the right cycle time/angle (8 min/7&#176;)</td>
		</tr>
		<tr>
			<td>No perfusion possible due to presence of air bubbles</td>
			<td>Remove air bubbles using a P20 or P200 pipette</td>
		</tr>
		<tr>
			<td>No lumens are formed, cells migrate as single cells</td>
			<td>Angiogenic sprouting mixture was added before a monolayer was formed</td>
			<td>Wait an additional 24 h before adding the angiogenic growth factors</td>
		</tr>
		<tr>
			<td>Major variation in sprouting density</td>
			<td>Differences in cell densities after seeding</td>
			<td>Check if cell density is homogenous and comparable between microfluidic units. Add another droplet of cell suspension if necessary</td>
		</tr>
	</tbody>
</table>
Table 1: Troubleshooting common errors.

Watch this Scientific Journal Video about Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro at JoVE.com

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

standardized-scalable-assay-to-study-perfused-3d-angiogenic-sprouting

Research

JoVE Journal

Bioengineering

30.4K Views.  Leiden University Medical Center. Deze in vitro test toont angiogeen ontkiemen in een hoge doorvoer en schaalbaar platform. Dit kan worden gebruikt om nieuwe doelen in angiogenese te vinden. Deze angiogenese test combineert complexe cellulaire micro-omgevingen met gradiënten en perfusie met een platform dat compatibel is met hoge doorvoer screening.Angiogenese speelt een belangrijke rol in zowel de gezondheid als de ziekte. Inzicht in de mechanismen die optreden tijdens angiogenese kan ons helpen om te begrijpen hoe k...

Video: Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Biology

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

Cancer Research

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

Developmental Biology

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Study of Angiogenesis Using Scratch Wound Migration and Spheroid Sprouting Assays

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

Author Spotlight: Investigating Angiogenesis Through Challenges and Innovations in Assay Development

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

Medicine

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

Versatile 2.5D Model for Analyzing Cellular Sprouting in Porcine Arteries

2.5D Model for Ex Vivo Mechanical Characterization of Sprouting Angiogenesis in Living Tissue

Sprouting angiogenesis is the formation of new blood vessels from pre-existing vasculature and is of great importance for physiological such as tissue growth and repair and pathological processes, including cancer and metastasis. The multistep process of sprouting angiogenesis is a molecularly and mechanically driven process. It consists of induction of cellular sprout by vascular endothelial growth factor, leader/follower cell selection through Notch signaling, directed migration of endothelial cells, and vessel fusion and stabilization. A variety of sprouting angiogenesis models have been developed over the years to better understand the underlying mechanisms of cellular sprouting. Despite advancements in understanding the molecular drivers of sprouting angiogenesis, the role of mechanical cues and the mechanical driver of sprouting angiogenesis remains underexplored due to limitations in existing models. In this study, we designed a 2.5D ex vivo model that enables us to mechanically characterize cellular sprouting from a porcine carotid artery using traction force microscopy. The model identifies distinct force patterns within the sprout, where leader cells exert pulling forces and follower cells exert pushing forces on the matrix. The model's versatility allows for the manipulation of both chemical and mechanical cues, such as matrix stiffness, enhancing its relevance to various microenvironments. Here, we demonstrate that the onset of sprouting angiogenesis is stiffness-dependent. The presented 2.5D model for quantifying cellular traction forces in sprouting angiogenesis offers a simplified yet physiologically relevant method, enhancing our understanding of cellular responses to mechanical cues, which could advance tissue engineering and therapeutic strategies against tumor angiogenesis.

Sprouting angiogenesis, fundamental for development and disease, involves complex molecular and mechanical processes. We present a versatile 2.5D ex vivo model that analyzes cellular sprouting from porcine carotid arteries, revealing stiffness-dependent angiogenesis and distinct leader-follower cell mechanics. This model aids in advancing tissue engineering strategies and cancer therapy approaches.

Sprouting angiogenesis is the formation of new blood vessels from pre-existing vasculature and is of great importance for physiological such as tissue ...

Bead Sprouting Assay for Early-Stage Angiogenesis Assessment: An In Vitro Bead-Based Model to Study Endothelial Sprouting in the Presence of Pericytes

Source: Azam, S. H. et al. <a href="https://www.jove.com/v/57309">Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay.</a> J. Vis. Exp. (2018).
This video demonstrates a modified microcarrier beads-based assay incorporating pericytes to study endothelial sprouting during the early stages of angiogenesis. The assay provides an in vitro experimental model to understand molecular mechanisms of angiogenesis and the effect of anti-angiogenetic therapeutics.

Source: Azam, S. H. et al. Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay. J. Vis. Exp. (2018).
This video demonstrates a modified ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

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