Name: stepwise cell seeding on tessellated scaffolds to study sprouting blood vessels
Uploaded: 2021-01-14T15:00:00
Description: Watch this Scientific Journal Video about Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels at JoVE.com

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.

1. Tessellated scaffold fabrication
NOTE: Photolithography is a widespread technique that requires specialized equipment typically housed within a nanofabrication facility/laboratory. The method laid out in this protocol was generalized as much as possible for the audience; however, slight changes to procedures may be necessary depending on equipment available to the reader. We recommend performing these procedures in a clean room at a nanofabrication facility&#160;to ensure the highest process ...

<ol>
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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;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&#34;, 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>
		</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" /> 
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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, ...

Our protocol allows researchers to study the vessel network formation process in a clear and easily trackable fashion, opening the doors to new studies and shedding light on blood vessel behavior. This technique presents the possibility of creating highly organized and repeatable vascular networks with respond to the surrounding environment. Endothelial cells from patients suffering from a vascular disease can be retrieved in using this system, making it possible to recreate the vascular disease and find an appropriate treatment.The proposed method can be extended for studying vascular network behavior when paired with different cell types, allowing to observe the interaction of developing vessels with a specific tissue type. Begin by submerging the scaffolds in 70%ethanol for a minimum of 15 minutes, then wash them twice in PBS. Mix 1.5 microliters of fibronectin stock solution with 28.5 microliters of PBS per scaffold to be seeded.Prepare one fibronectin dilution to be used for all scaffolds to avoid pipetting errors. Place the scaffolds sparsely on top of a hydrophobic surface and cover each scaffold with 30 microliters of the fibronectin dilution. Replace the plate's lid and put the fibronectin-covered scaffolds into an incubator set to 37 degrees Celsius and 100%humidity for a minimum of one hour.After incubation, lightly rinse the scaffolds in PBS to remove fibronectin remnants. The scaffolds can be kept in PBS at four degrees Celsius for up to a week. Prepare endothelial cell or EC medium by mixing the basal medium with the corresponding medium kit components, including an antibiotic solution, FBS, and endothelial cell growth supplements as indicated by the manufacturer.Make the human adipose microvascular endothelial cell suspension in EC medium with a concentration of four million cells per milliliter. Using forceps, place one fibronectin-coated scaffold per well in a non-TC 24-well plate. Cover each scaffold with 25 microliter droplets of the cell suspension, making sure not to let the suspension flow away from the scaffold.Put the lid on the plate and place it in an incubator at 37 degrees Celsius, 5%carbon dioxide and 100%humidity for 60 to 90 minutes. After the incubation, fill each well with 700 microliters of EC medium. Incubate the endothelialized scaffolds until EC confluence can be observed using fluorescent microscopy or for three days.Change the medium every other day. Prepare DPSC medium by mixing 500 milliliters of low glucose DMEM, 57.5 milliliters of FBS, 5.75 milliliters of non-essential amino acids, 5.75 milliliters of GlutaMAX, and 5.75 milliliters of penicillin-streptomycin-nystatin solution. Transfer the endothelialized scaffolds into a new non-TC 24-well plate.Discard all media from the current plate using a pipette or vacuum suction, taking care not to apply vacuum directly to the scaffold. Place one scaffold into the center of each well, then dry the surrounding area with light vacuum. Dilute thrombin and fibrinogen stock solutions with PBS to a final concentration of five units per milliliter and 15 milligrams per milliliter respectively.Prepare an eight million DPSC per milliliter suspension in the thrombin dilution and distribute 12.5 microliters of the suspension into individual microtubes per scaffold to be seeded. Set a five to 50 microliter pipette to 12.5 microliters and fill it with fibrinogen solution. Without removing the tip, set the pipette to 25 microliters.The material in the tip should rise and leave an empty volume. Slowly press the plunger button until the liquid reaches the tip opening, but does not leak out. Hold the plunger in this position and put the tip into one of the microcentrifuge tubes containing the cells in thrombin suspension, making sure the tip is in contact with the liquid.Gently release the plunger button and draw the cell suspension into the tip. Thoroughly mix both materials, avoiding bubble formation. Quickly dispense the mixed materials on top of an endothelialized scaffold.Repeat the previous steps for each scaffold, making sure to change tips between uses to avoid unexpected fibrin gel formation within the tip. Replace the plate lid and incubate the scaffolds at 37 degrees Celsius, 5%carbon dioxide and 100%humidity for 30 minutes. After incubation, fill each well with one milliliter of one-to-one DPSC and EC medium.Culture for one week, changing the medium every other day. At different time points during culture, remove the medium from the well and image the constructs using a confocal microscope to study the vascular development or any other parameter of interest. This protocol allows for the fabrication of tessellated scaffolds made of SU-8 photoresist.Scaffolds with distinct compartment geometries and highly accurate and repeatable features were obtained. With traditional simultaneous seeding of both endothelial cells and support cells, the cells were homogeneously distributed over the scaffold resulting in unpredictable and disorganized vascular networks. Contrarily, stepwise cell seeding resulted in highly organized vascular networks.When using fluorescent endothelial cells, the vessels can be imaged in real time. Red fluorescent protein expressing endothelial cells were cultured on hexagonal scaffolds and imaged. The support cells were added at day one and the vascular networks were imaged every other day to quantify vessel development.For each time point, wide images of the whole scaffold were taken. Vessel growth was quantified as total vessel length and area. A confocal imaging time-lapse was performed to allow single vessel tracking using multi-colored endothelial cells.The vessel path was generated from the time-lapse, making it possible to observe vessel migration. Vessel maturation was observed by the presence of smooth muscle actin and support cells. For the circular, hexagonal, and squared compartments, the cells multiply and organize into structures.By day seven, all shapes showed a rich and complex vascular network. Higher magnification images revealed a denser smooth muscle actin and support cell presence co-localized with formed vessels, evidencing support cell recruitment and differentiation surrounding vascular structures. This technique can be used to study the behavior of three-dimensional vascular networks when exposed to different conditions, such as specific cell inhibitors or in the presence of additional cell types.

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