This research was supported by funding from the University of Michigan - Israel Partnership for Research. The authors would like to thank Uri Merdler, Lior Debbi&#160;and Galia Ben David for their great assistance and support, Nadine Wang, Ph.D. and Pilar Herrera-Fierro, Ph.D. of the Lurie Nanofabrication Facility at the University of Michigan, as well as Luis Solorio, Ph.D. for enlightening discussions of photolithography techniques.

<ol>
	<li>Novosel, E. C., Kleinhans, C., Kluger, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+is+the+key+challenge+in+tissue+engineering.">Vascularization is the key challenge in tissue engineering.</a> Advanced Drug Delivery Reviews. 63 (4), 300-311 (2011).</li><li>Landau, S., Guo, S., Levenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+Engineered+Vasculature+within+3D+Tissue+Constructs.">Localization of Engineered Vasculature within 3D Tissue Constructs.</a> Frontiers in Bioengineering and Biotechnology. 6, 2 (2018).</li><li>Griffith, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+Limits+of+an+in+Vitro+Thick+Prevascularized+Tissue.">Diffusion Limits of an in Vitro Thick Prevascularized Tissue.</a> Tissue Engineering. 11 (12), (2005).</li><li>Potente, M., Gerhardt, H., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+and+therapeutic+aspects+of+angiogenesis.">Basic and therapeutic aspects of angiogenesis.</a> Cell. 146 (6), 873-887 (2011).</li><li>Landau, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tropoelastin+coated+PLLA-PLGA+scaffolds+promote+vascular+network+formation.">Tropoelastin coated PLLA-PLGA scaffolds promote vascular network formation.</a> Biomaterials. 122, 72-82 (2017).</li><li>Lesman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vessel-like+networks+within+multicellular+fibrin-based+constructs.">Engineering vessel-like networks within multicellular fibrin-based constructs.</a> Biomaterials. 32 (31), 7856-7869 (2011).</li><li>Richards, D., Jia, J., Yost, M., Markwald, R., Mei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Bioprinting+for+Vascularized+Tissue+Fabrication.">3D Bioprinting for Vascularized Tissue Fabrication.</a> Annals of Biomedical Engineering. 45 (1), 132-147 (2017).</li><li>Levenberg, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vascularized+skeletal+muscle+tissue.">Engineering vascularized skeletal muscle tissue.</a> Nature Biotechnology. 23 (7), 879-884 (2005).</li><li>Rouwkema, J., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+and+Angiogenesis+in+Tissue+Engineering:+Beyond+Creating+Static+Networks.">Vascularization and Angiogenesis in Tissue Engineering: Beyond Creating Static Networks.</a> Trends in Biotechnology. 34 (9), 733-745 (2016).</li><li>Miller, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+casting+of+patterned+vascular+networks+for+perfusable+engineered+three-dimensional+tissues.">Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues.</a> Nature Materials. 11, (2012).</li><li>Gariboldi, M. I., Butler, R., Best, S. M., Cameron, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vasculature+Architectural+effects+on+microcapillary-like+structure+self-assembly.">Engineering vasculature Architectural effects on microcapillary-like structure self-assembly.</a> PLOS ONE. 14 (1), 1-13 (2019).</li><li>Blache, U., Guerrero, J., G&#252;ven, S., Klar, A. 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The authors have nothing to disclose.

The need for a rich vasculature within embedded in engineered tissues is critical for construct survival and proper function1. Although engineering the vascular system has been the focus of a vast amount of research, much is left to investigate and understand24. In particular, when recreating a specific tissue, the microvasculature should behave and organize accordingly12. The most common approach for microvessels generation is co-seeding endothelial and support cells within a suitable 3D environment compatible for cell attachment, proliferation, and vessel formation25. This methodology often results in greatly unorganized networks, making it difficult to study the microvascular behavior22. Here, the presented protocol provides a new tool that generates highly organized vascular networks in a high-throughput system, with vessels that can be easily tracked and monitored through time, to study their development and behavior. The stepwise seeding of the ECs (step 2-3), followed by the SCs (step 4), are critical steps to achieve such organized networks22. Additionally, this technique presents a real 3D environment for vascular models, in which vessels can migrate in the three dimensions and create relevant structures. In contrast, more popular methods for vascular modelling, such as microfluidics systems, only offer a 2.5D tissue representation26.
The proposed method can be easily modified and applied to study many different factors affecting the vessel network development and behavior. Photolithographic SU-8 resin scaffold fabrication is a common technique with an impressive versatility that enables creating a wide variety of shapes26,27, with the potential to design structures resembling complex native constructs, such as the alveoli or the nephrotic unit. Nonetheless, a drawback of using SU-8 is its low bioabsorbability27, making the platform mainly a research tool, instead of an implantable tissue. However, this could be improved by utilizing biocompatible materials which can crosslink under UV/visible light illumination, and 3D printed using accurate techniques like stereolitography29. The resulting vascularized constructs could then be implanted in an animal model30. Another limitation of the system is the scaffold maximum achievable thickness with high detail accuracy, limited by the stereolithographic technique31. The proposed protocol is suitable for creating thin scaffold which might represent native tissue sizes.
This platform offers the potential to investigate several variables affecting the vascularization phenomena4,32. First, the interactions between different types of ECs and SCs, and their vessel formation, development and maturation capabilities, which are known to differ when using cells from different tissue origins33. Second, the effect of growth factors, small molecules, and inhibitors on the vasculature, allowing for a clear visualization of their impact in real time34,35. Third, the addition of other cell types, spheroids, or organoids and their communication and interaction with the forming vessels36. For this, the proposed system represents a powerful tool for investigating the microvascular system.
Future steps will take advantage of the proposed system to investigate the effect of mechanical stimuli, such as interstitial flow and mechanical stretching37,38, on the developing vasculature. This will hopefully shed light on new aspects that will expand the current knowledge and state-of-the-art research of the vascular mechanobiology.

The field of tissue engineering is rapidly progressing towards the fabrication of engineered constructs to replace missing or damaged organs and tissues1. However, fully functional constructs have yet to be achieved, in part, since generating operational vascular networks for tissue nourishment remains an outstanding challenge. Without proper vascularization, engineered tissues are limited to a passive diffusion transport of oxygen and nutrients, constraining the maximum viable tissue thickness to the diffusion limit, approximately 200 &#181;m2. Such thicknesses are not suitable to repair large tissue defects or for full organ fabrication, which renders the presence of functional vascular network a mandatory characteristic for functional and implantable tissues3.
The vascular system is comprised of a wide variety of blood vessels, with different sizes, phenotypes, and organization, tightly related to the host tissue. Understanding the behavior, response and migration decisions made by the developing and sprouting vessels can instruct their integration in engineered tissues4. Currently, the most common approach for creating in vitro vascular networks is combining endothelial cells (ECs) with support cells (SCs, with the capability to differentiate into mural cells), seeded within a three-dimensional micro-environment. This environment provides chemical and physical cues to allow the cells to attach, proliferate and self-assemble into vessel networks2,5,6,7,8. When co-cultured, SCs secrete extracellular matrix (ECM) proteins while providing mechanical support to the ECs, which form the tubular structures. Furthermore, a cross-interaction between both cell types promote tubulogenesis, vessel sprouting and migration, in addition to the SCs maturation and differentiation into &#945;-smooth muscle actin-expressing (&#945;SMA) mural cells4. Vessel network development is most commonly studied in 3D environments created using hydrogels, porous polymeric scaffolds, or a combination thereof. The latter option equally provides a cell-friendly environment and the required mechanical support for both the cells and the ECM9.
A great amount of work has been carried out to study vascular development, including co-culturing the cells on hydrogels10, hydrogels-scaffold combinations11,12, 2D platforms, and microfluidic devices13. However, hydrogels can be easily deformed by the cell-exerted forces14, while 2D and microfluidics systems fail to recreate a closer-to-nature environment to obtain a more extrapolatable response15,16. Understanding how forming vessels react to their surrounding environment can provide critical insight that might allow for the fabrication of engineered environments with the capability of guiding the vessel development in a predictable manner. Understanding vascular formation phenomena is especially critical to keep pace with the rapid emergence of submicron-to-micron scale fabrication techniques, such as stereolithography, digital projection lithography, continuous liquid interface production, 3D melt-electro jetwriting, solution based 3D electro jet writing, and emerging bioprinting techniques17,18,19,20,21. Aligning the control of these micromanufacturing techniques with a deepened understanding of vascular biology is key to the creation of an appropriate engineered vasculature for a target tissue.
Here, we present a 3D system to study the response of new forming and sprouting vessels to the surrounding scaffold geometry, observing their sprout origin and subsequent migration22. By utilizing 3D scaffolds with tessellated compartment geometries, and a two-step seeding technique, we succeeded to create highly organized vascular networks in a clear and easy to analyze fashion. The tessellated geometries provide a high throughput system with individual units containing vessels that respond to their local environment. Using multicolored ECs, we tracked sprout formation origins and subsequent migration patterns, correlated to the compartment geometry and the SCs location22.
Although the proposed protocol has been prepared to analyze the effects of geometrical cues on vascularization behavior, this approach can be expanded and applied to a variety of new applications. The tessellated scaffold and the easily imageable networks allow for the straightforward analysis of different ECs and SCs interaction, the addition of specific organ cells and their interaction with the vascular networks, drug effect on vascular networks, and more. Our suggested system results very versatile and of simple fabrication and processing.

<table><tbody><tr><td>Angiotool freeware</td><td>NIH-CCR</td><td></td><td>Free download at https://ccrod.cancer.gov/confluence/display/ROB2/Home</td></tr><tr><td>Bovine albumin serum Probumin</td><td>Millipore</td><td>82-045-1</td><td></td></tr><tr><td>Dental pulp stem cells</td><td>Lonza</td><td>PT-5025</td><td></td></tr><tr><td>ECM media + bullet kit</td><td>Sciencell</td><td>#1001</td><td></td></tr><tr><td>Ethanol 96%</td><td>Gadot-Group</td><td>64-17-5</td><td></td></tr><tr><td>Evicel fibrin sealant</td><td>Johnson&amp;amp;Johnson</td><td>EVB05IL</td><td>Provides both thrombin and fibrinogen (BAC2) solutions</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050061</td><td></td></tr><tr><td>Goat anti-mouse Cy3 antibody</td><td>Jackson</td><td>115-166-072</td><td></td></tr><tr><td>Goat anti-rabbit Alexa-Fluor 488</td><td>Thermo- Fisher Scientific</td><td>A11034</td><td></td></tr><tr><td>Human adipose microvascular cells</td><td>Sciencell</td><td>#7200</td><td></td></tr><tr><td>Human fibronectin</td><td>Sigma</td><td>F0895-5MG</td><td>Stock concentration: 1 mg/mL</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>Free download at https://imagej.nih.gov/ij/download.html</td></tr><tr><td>Isopropyl alcohol</td><td>Gadot-Group</td><td>67-63-0</td><td></td></tr><tr><td>Lift-off reagent</td><td>Kayaku Advanced Materials, Inc</td><td>G112850</td><td>Commercial name Omnicoat</td></tr><tr><td>Low-glucose DMEM</td><td>Biological Industries</td><td>01-050-1A</td><td></td></tr><tr><td>Mouse anti-SMA antibody</td><td>Dako</td><td>M0851</td><td></td></tr><tr><td>NEAA</td><td>Gibco</td><td>11140068</td><td></td></tr><tr><td>Paraformaldehyde solution 4% in PBS</td><td>ChemCruz</td><td>SC-281692</td><td></td></tr><tr><td>Penicillin-Streptomycin-Nystatin Solution</td><td>Biological Industries</td><td>03-032-1B</td><td></td></tr><tr><td>Phospate buffered saline (PBS)</td><td>Sigma</td><td>P5368-10PAK</td><td></td></tr><tr><td>Rabbit anti-vWF antibody</td><td>Abcam</td><td>ab9378</td><td></td></tr><tr><td>Silicon wafer</td><td>Silicon Valley Microelectronics (SVM)</td><td></td><td>Wafers 4&quot;, Type N-1-10, 500-550 microns thick</td></tr><tr><td>SU-8 2050 photoresist</td><td>Kayaku Advanced Materials, Inc</td><td>Y11058</td><td></td></tr><tr><td>SU-8 developer</td><td>Kayaku Advanced Materials, Inc</td><td>Y020100</td><td></td></tr><tr><td>Tryton-X 100</td><td>BioLab LTD</td><td>57836</td><td></td></tr></tbody></table>

stepwise cell seeding on tessellated scaffolds to study sprouting blood vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

The presented protocol, using stereolithography techniques, allows for the fabrication of tessellated scaffolds made of SU-8 photoresist. Scaffolds with distinct compartment geometries (squares, hexagons, and circles), and highly accurate and repeatable features were obtained (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61995/61995fig01.jpg" /> 
Figure 1:&#160;Representative scanning electron microscopy images of the tessellated square, circular, and hexagonal scaffold geometries (scale bar = 500 &#181;m).&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61995/61995fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
With a stepwise cell seeding (steps 2 to 4), the fabricated scaffolds were used to create highly organized vascular networks. When using a traditional simultaneous seeding of both ECs and SCs, the resulting vessels lacked a clear organization. For this, the scaffold fibronectin coating was performed (step 2), the scaffold endothelialization step was skipped (step 3), and the DPSC and HAMEC were simultaneously co-seeded in fibrin gel (step 4). In this fashion, the cells are homogeneously distributed over the scaffold (Figure 2, top row), resulting in unpredictable and disorganized developed vascular networks that do not seem to interact with the surrounding scaffold. Contrarily, firstly seeding the ECs on the scaffold walls provides an accurate initial endothelial cell patterning. The later addition of SCs within a fibrin gel results in a predictable tubulogenesis phenomenon, with forming vessels closely following the shape of the scaffold wall, and sprouting new vessels migrating into the compartment space (Figure 2, bottom row).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61995/61995fig02.jpg" /> 
Figure 2: Vascularization comparison between simultaneous vs. stepwise cell seeding.&#160;Representative images of vascular development in tessellated scaffolds for a simultaneous (top row) cell seeding of ECs (red) and SCs (green), and step-wise cell seeding (bottom row) at days 1 and 5. The stepwise seeding results in organized vascular networks that follow the scaffold walls and sprout into the compartment space (scale bar: 100 &#181;m).&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61995/61995fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
When using fluorescent ECs, either transfected or dyed, the vessels can be imaged in real time without the need to fix and terminate the experiment for each time point. Red fluorescent protein expressing ECs (RFP-ECs) were cultured on hexagonal scaffolds and imaged after seeding (Figure 3A, day 0). At day 1, the SCs were added and the vascular networks were imaged every other day to quantify the vessel development (Figure 3A, days 1, 3, 5 and 7). For each time point, wide images of the whole scaffold were taken (Figure 3B). For every compartment, the vessels mainly organized and interacted with cells located within their confinement. Hence, each compartment was isolated and the superfluous vessels outside the compartment were removed using ImageJ. The clean, single-compartment images were then analyzed using Angiotool. Angiotool returned a spread sheet file containing several vessel parameters, and a visual representation of the main network characteristics, such as skeleton, intersection points, and vessel surface. The obtained data was analyzed using the statistical analysis software Prism, and a clear vessel growth was observed for total vessel length and area during the experiment time frame (Figure 3C). During a 1-week experiment, vessels are expected to further develop and extended as shown in Figure 3A and Figure&#160;2C. Decreasing vessel length or area, failure to form vessels by day 3 or vessels forming as shown in the top row of Figure 2 can be interpreted as a failed experiments.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61995/61995fig03.jpg" /> 
Figure 3: Representative development images and analysis of organized vascular networks.&#160;(A) ECs (red) reach confluence on the scaffold wall on which SCs are afterwards seeded; the SCs addition represents day 0 of the experiment. At day 1 after the SCs seeding, the ECs detach to the compartment space and start forming vessels that will continue sprouting and connecting at further days. (B) Confocal image processing steps for vascular network analysis (i) A wide confocal image containing several compartments is taken, (ii) a single compartment is cropped (demarked by the white dashed hexagon), (iii) then the vascular network channel is separated, and all vessels outside the compartment walls are cropped out. The single compartment image is analyzed using Angiotool, returning a list of vascular parameters complemented with visual markers, such as the vessel area (outlined in yellow), the vessels length (displayed with green lines), and the intersection points (marked as blue dots). (C) Comparative results of the total vessel length and the total vessel area within hexagonal compartments at different time points (results are presented as mean &#177; SD, n &#62; 6; all scale bars: 200 &#181;m).&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61995/61995fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Using multicolored ECs to facilitate single cell identification, a confocal imaging time lapse was performed to allow single vessel tracking (Supplementary Video 1). The vessels were observed and tracked using the Manual Tracking ImageJ plugin. The tip cell was selected for each frame of the movie (Figure 4A) until the vessel anastomosed with the surrounding vasculature. As a result, the Manual Tracking plugin generated the vessel path in real time, which allowed to observe the vessel migration (Figure 4B).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61995/61995fig04.jpg" /> 
Figure 4: Representative sprouting vessel tracking.&#160;(A) A multicolored ECs (green, red, and blue) time lapse is used to facilitate single vessel identification. A sprouting vessel is identified and tracked using the ImageJ plugin&#160;Manual Tracking. The end side of the vessel is marked for every time point to track in real time; the black-in-white marker was added to show the selected vessel end point. (B) The resulting 2D tracking of the vessel, as processed by the ImageJ plugin, showing the farthest point with a dot, and the formed path with a line (scale bar = 200 &#181;m).&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61995/61995fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Vessel maturation, represented by the presence of SMA+ SCs, can be easily observed in the proposed platform. Vasculatures presenting higher numbers of SMA+ SCs represent a more mature network, since SMA expression correlates with vessel stabilization over time23. For the circular, hexagonal and squared compartments, the amount of SMA+ SCs increases over time (Figure 5A). By day 3, all shapes showed scattered SMA+ SCs and uncomplex vessels with few or no sprouts whatsoever. By day 7, all shapes showed a rich and complex vascular network, with a higher presence of SMA+ SCs surrounding the vessels. Furthermore, higher magnification images reveal a denser SMA+ SCs presence co-localized with formed vessels, evidencing the SCs recruitment and differentiation surrounding vascular structures (Figure 5B).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61995/61995fig05.jpg" /> 
Figure 5: SMA+ SCs and blood vessels increase over time.&#160;A) Smooth muscle actin (red) and vWF (vessels, green) are shown for vascular networks in circular, squared and hexagonal compartments at day 3 and day 7. Both vasculature extension and SMA-expressing support cells (SMA+ SCs) increase over time, signifying a higher vessel maturation and complexity (scale bar = 200 &#181;m).&#160;B) Representative images of the SMA+ SCs denser accumulation around vessels at day 7. The nuclei (blue) in the composite image reveal the presence of SCs not expressing the SMA protein (scale bar = 50 &#181;m).&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61995/61995fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Video 1: multicolored ECs time lapse for vessel migration tracking.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61995/Supplementary Video 1.mp4" target="_blank">Please click here to download this video.</a>

Watch this Scientific Journal Video about Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels at JoVE.com

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

stepwise-cell-seeding-on-tessellated-scaffolds-to-study-sprouting

Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.4K Views.  Technion. Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

Video: Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

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

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 ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

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.

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 ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

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 ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

Summary

Explore More Videos

Chapters in this video

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

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

Micropatterning and Assembly of 3D Microvessels

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

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Microfluidic Model to Mimic Initial Event of Neovascularization

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

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

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