eoe sub articles

EoE Sub Articles

1. Preparing Beads for Gel Implantation
<ol>
	<li>Take a culture containing microcarrier beads coated with Human Umbilical Vein Endothelial Cells (HUVEC) and pericytes.</li>
	<li>Examine the dishes containing the microcarrier-coated beads under the microscope using the 20X objective to ensure that all beads have been sufficiently coated by endothelial cells.</li>
	<li>Vigorously pipette the plated solution of cells to detach them from the 6-cm plate and place the solution into a tube. Wash the plate 2X additional times with complete media and transfer to the tube to collect all residual beads that may be adhered to the plate.
	<ol>
		<li>Avoid introducing air bubbles and use a p1000 pipette to minimize shear stress on the endothelial cell-coated beads.</li>
	</ol>
	</li>
	<li>Wait 3 min to allow the beads in the solution to settle to the bottom of the tube.</li>
	<li>Remove as much of the supernatant as possible without disturbing the bead pellet using a p1000 pipette tip (do not use a vacuum aspirator).</li>
	<li>Add 1 mL of complete media to each tube and resuspend the beads.</li>
	<li>Wait 30-60 s to allow the beads to settle to the bottom. Then remove as much of the supernatant as possible using a P1000 pipette without disturbing the bead pellet. Do not use a vacuum aspirator as the likelihood of accidentally removing the bead pellet is greatly increased.</li>
	<li>Repeat Steps 1.6 and 1.7 two additional times. The goal is to remove as much of the free-floating HUVEC that may have been detached from the 6-cm plate as possible, while not removing too many HUVEC-coated beads, which should settle in the round-bottomed Fluorescence Activated Cell Sorting (FACS) tubes more quickly than free-floating cells.</li>
	<li>Add 1 mL of complete media to the beads.</li>
</ol>
2. Embedding Coated Beads in a Fibrin Gel
<ol>
	<li>Remove as much media as possible without disturbing the bead pellet in each FACS tube using a P1000 pipette.</li>
	<li>Resuspend the beads in 2.5 mL of fibrinogen solution.</li>
	<li>Pipette 13 &#181;L of thrombin solution into each desired well of a 24-well glass-bottomed plate. Plating in a glass-bottomed plate is essential for enabling optimal imaging of the sprouts. 
	NOTE: Do not leave thrombin sitting in a well for more than 5-10 min before the next step.</li>
	<li>Add 0.5 mL of bead/fibrinogen solution to each well.
	<ol>
		<li>Be sure to resuspend the bead solution well prior to pipetting the solution each time.</li>
		<li>Take caution to carefully pipette directly into the thrombin already present in the well. Slowly pipette up and down 2-3 times to thoroughly mix the solution, taking caution to not introduce any air bubbles.</li>
		<li>Be sure to change the pipette tip between wells.</li>
		<li>Take caution to not move or disturb the plate at any point during initial fibrin clotting, as any movement may disrupt the gel formation.</li>
	</ol>
	</li>
	<li>Leave the plate sitting in the hood for 30 min at room temperature.</li>
	<li>Carefully transfer the plate to a 37 &#176;C incubator for an additional 1.5-2 h to allow the gel to fully solidify</li>
</ol>
3. Plating Fibroblasts on Top of the Fibrin Gel
<ol>
	<li>During the last 30 min of the gel solidifying, detach Normal Human Lung Fibroblasts (NHLF) as follows:
	<ol>
		<li>Wash a T175 flask of cells with 10 mL of Phosphate Buffered Saline (PBS), then add 5 mL of cell detachment solution and incubate at 37 &#176;C for 10 min.</li>
		<li>Resuspend NHLF at a concentration of 20,000 cells/mL in complete media. 
		NOTE: NHLF cells can be cultured with Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin Streptomycin.</li>
	</ol>
	</li>
	<li>Plate 1 mL of the NHLF cell suspension onto the gel of each well and place it back in the incubator.</li>
	<li>Change complete media every other day for the duration of the assay.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sterile Pipette tips</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EGM2 Media Bullet Kit</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>HUVEC Media</td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Gibco</td>
			<td>11095114</td>
			<td>10T1/2 Media</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965118</td>
			<td>NHLF Media</td>
		</tr>
		<tr>
			<td>Tissue culture-grade PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Magnesium and calcium free</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td>For lifting HUVEC</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies</td>
			<td>15050065</td>
			<td>For lifting 10T 1/2 and NHLF</td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>Lonza</td>
			<td>C2517A</td>
			<td></td>
		</tr>
		<tr>
			<td>10T 1/2</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NHLF</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytodex 3 microcarrier beads</td>
			<td>Sigma</td>
			<td>C3275</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture-coated 6 and 10 cm plates</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen from bovine plasma</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>t9549</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>a3428</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Tubes</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture incubator and hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well glass bottom plates</td>
			<td>MatTek</td>
			<td>P24G1.513F</td>
			<td>Glass-bottom plates are needed only if the sprouts are going to be imaged. If not, tissue culture plastic is also acceptable.</td>
		</tr>
	</tbody>
</table>

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.

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. Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay. J. Vis. Exp. (2018).
This video demonstrates a modified ...

Angiogenesis is the formation of new blood vessels from the existing blood vessels during developmental and pathological conditions. Extensive communication between the endothelial cells that line the inner surface of blood vessels and the surrounding pericytes - supporting cells on the outer side of blood vessels - is crucial for angiogenesis.
To study in vitro angiogenesis, begin with endothelial cells and pericytes-coated microcarrier beads suspended in a fibrinogen solution - a soluble glycoprotein that acts as a fibrin precursor.
Now, pipette the bead suspension into a culture plate containing a solution of thrombin - a proteolytic enzyme, and incubate. Thrombin binds to the fibrinogen molecule, causing its cleavage to form insoluble fibrin monomers, forming polymeric fibrin gel. This step ensures the embedding of cell-coated beads in the fibrin gel.
Next, plate a culture of fibroblasts - connective tissue precursor cells - on top of the fibrin gel and incubate to allow fibroblasts&#39; activation.
During incubation, the fibroblasts secrete crucial angiogenic-growth factors that trigger endothelial cell activation. In combination, adherent pericytes and endothelial cells secrete signaling molecules that help in endothelial cell sprouting in the fibrin gel.
The endothelial sprouts appear as pericyte-wrapped elongated cell protrusions, depicting the early stage of angiogenesis.

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249 Views. Source: Azam, S. H. et al. Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay. 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. 

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

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

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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 ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

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

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