Name: the aortic ring co-culture assay: a convenient tool to assess the angiogenic potential of mesenchymal stromal cells in vitro
Uploaded: 2017-09-18T15:00:00
Description: Watch this Scientific Journal Video about The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro at JoVE.com

The authors thank the following staff members and research personnel: Andr&#233;e Gauthier-Fisher, Matthew Librach, Tanya Barretto, Tharsan Velauthapillai, and Sarah Laronde.

All studies involving animals were conducted and reported according to ARRIVE guidelines29. All studies were performed with institutional research ethics board approval (REB number 4276). All animal procedures were approved by the Animal Care Committee of the University Health Network (Toronto, Canada), and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, 8th edition (National institutes of Health 2011).
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<ol>
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D., Laschke, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geraniol+Suppresses+Angiogenesis+by+Downregulating+Vascular+Endothelial+Growth+Factor+(VEGF)/VEGFR-2+Signaling.">Geraniol Suppresses Angiogenesis by Downregulating Vascular Endothelial Growth Factor (VEGF)/VEGFR-2 Signaling.</a> PLoS One. 10 (7), e0131946 (2015).</li><li>Masson, V. V., 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=Mouse+Aortic+Ring+Assay:+A+New+Approach+of+the+Molecular+Genetics+of+Angiogenesis.">Mouse Aortic Ring Assay: A New Approach of the Molecular Genetics of Angiogenesis.</a> Biol Proced Online. 4, 24-31 (2002).</li><li>Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mesenchymal+stem+cells+for+tissue+engineering+versus+regenerative+medicine.">Adult mesenchymal stem cells for tissue engineering versus regenerative medicine.</a> J Cell Physiol. 213 (2), 341-347 (2007).</li><li>Caplan, A. I., Dennis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+as+trophic+mediators.">Mesenchymal stem cells as trophic mediators.</a> J Cell Biochem. 98 (5), 1076-1084 (2006).</li><li>Keating, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells:+new+directions.">Mesenchymal stromal cells: new directions.</a> Cell Stem Cell. 10 (6), 709-716 (2012).</li><li>Kilroy, G. E., 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=Cytokine+profile+of+human+adipose-derived+stem+cells:+expression+of+angiogenic,+hematopoietic,+and+pro-inflammatory+factors.">Cytokine profile of human adipose-derived stem cells: expression of angiogenic, hematopoietic, and pro-inflammatory factors.</a> J Cell Physiol. 212 (3), 702-709 (2007).</li><li>Tang, Y. L., 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=Paracrine+action+enhances+the+effects+of+autologous+mesenchymal+stem+cell+transplantation+on+vascular+regeneration+in+rat+model+of+myocardial+infarction.">Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction.</a> Ann Thorac Surg. 80 (1), 229-236 (2005).</li><li>Caplan, A. 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There are several critical stages in setting up a successful aortic ring assay MSC co-culture experiment. First, the most important steps when isolating and sectioning aorta are: 1) obtaining exclusively the thoracic segment of aorta; 2) carefully removing branching blood vessels, connective and adipose tissue and; 3) cutting even sections of the aorta (~1 mm) to limit variability between each assay. Second, the successful embedding of aortic rings into BME is critical for this assay. If the BME is not completely polymer...

The complex process of angiogenesis improves and maintains tissue perfusion by promoting new blood vessel development from pre-existing vasculature1. It is a tightly regulated, balanced process by pro-angiogenic and anti-angiogenic factors. Any deficiency in this system may lead to insufficient vessel maintenance or growth, causing severe ischemic diseases including myocardial disease, stroke, and neurodegenerative disorders. However, exaggerated vascular development is characteristic for conditions including cancer and inflammatory disorders2.
Developing therapies that aim to control angiogenesis to achieve favorable tissue regeneration is of key importance. Despite extensive preclinical and clinical investigations, attempts to stimulate angiogenesis using pro-angiogenic factors and microRNAs have failed to achieve desired outcomes3,4,5. Possible reasons for the transient effects include: limited longevity of angiogenic proteins and nucleic acids, and the finite number of targeted growth factors6,7. Although soluble angiogenic factors are essential for initiating angiogenesis, the maintenance and functionality of vasculature depend on supporting cell types including pericytes and smooth muscle cells8. The field of pro-angiogenic therapies is now exploring potential stem cell and progenitor cell sources that may provide angiogenic factors locally, while physically supporting newly developed vasculature, self-renewing or even differentiating into endothelial-like cells9,10. Finding the optimal angiogenic cell types with the capability to fulfill these functional requirements holds a great promise for ischemic tissue regeneration.
In order to successfully translate potential cell-based therapies into clinical trials, pre-clinical studies need to demonstrate their efficacy and highlight the underlying angiogenic mechanisms. Despite the high number of established angiogenesis assays, the field lacks a &#34;gold-standard&#34; in vitro assay that could reliably evaluate the efficacy of potential candidate cell types11,12,13. Most in vitro angiogenesis assays (including the endothelial proliferation, migration and tube formation assays) typically assess the effects of cells or compounds on endothelial cells&#39; phenotypical changes or differentiation into tubular and network structures14,15. While these features are critical for angiogenesis, a &#34;translatable&#34; assay should also evaluate: 1) the augmentation or replacement of the supporting cell types including pericytes or smooth muscle cells, 2) the processing of ECM and/or basement membrane, and 3) the efficiency to promote the formation of functional microvasculature. In vivo angiogenesis models, including the corneal assay and Matrigel plug assay, recapitulate the unique in vivo microenvironment but are challenged by the difficulty of tracking administered cells to observe physical interactions. Furthermore, in in vivo models, xeno-immune rejection can occur while testing potential allogeneic cell therapy candidates16. Ex vivo angiogenesis models, particularly the aortic ring assay can provide: 1) easy observation and quantification of tubular structures, 2) accessory supportive cells, 3) ECM from host and artificial supplies, 4) exclusion of inflammatory components, and 5) quick and inexpensive setup17,18. Typically, the aortic ring assay can test the angiogenic potential of small secretory proteins, pharmacological agents, and transgenic rodent models19,20,21.
MSCs are promising candidates for vascular regeneration primarily through their paracrine-mediated effects22,23,24. MSCs have been shown to secrete key angiogenic factors including Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), Insulin-like Growth Factor-1 (IGF-1), basic Fibroblast Growth Factor (bFGF), and angiopoeitin-1 (Ang-1)25,26. MSCs can also detect and home to ischemic or inflamed tissues, however, the exact mechanisms are still under investigation. Increasingly, the literature supports the hypothesis that most MSCs arise from perivascular cells, co-express pericyte markers, and can behave like pericytes27. HUCPVCs are a young source of MSCs derived from the perivascular region of the human umbilical cord. They represent a population of MSCs with pericyte-like properties and have been characterized from both FTM and term umbilical cords. FTM HUCPVCs demonstrate a high expression of pericyte markers including CD146 and NG2, high proliferative and multilineage potential, immune-privileged properties, and display a robust paracrine profile28. FTM HUCPVCs are an ideal candidate cell type to promote regeneration of injured tissue through the promotion of new vasculature via their pericyte-like properties.
To test the angiogenic potential and pericyte-like properties of human MSCs, a very limited number of angiogenesis assays are available where positive angiotropic migration (hereafter referred to as &#34;homing&#34;), ECM processing, and development of physical interactions between cell types can be investigated, while obtaining quantitative data on microvasculature development.
Hereby we present a protocol that describes a novel application of the aortic ring assay. Human MSCs were co-cultured with developing rat-derived aortic endothelial networks to assess their contribution to tube formation, maturation, and homeostasis. This version of the aortic ring assay assesses the ability and potency of cell therapy candidates to home to sites of angiogenesis, perform and mediate ECM processing, and contribute to endothelial tubular development through establishing pericyte-like physical interactions. In addition to quantifying the net effect of MSCs on in vitro endothelial network formation and observing intercellular interactions, we also optimized a protocol to isolate MSCs from co-cultures. By performing flow cytometry and qPCR, it is possible to characterize changes in MSC phenotype and gene expression following co-culture. As model cell types, we compared ontogenetically early (prenatal) and late (adult) sources of human MSCs: FTM HUCPVCs and human bone marrow-derived MSCs (BMSC), respectively, in the aortic ring assay. We propose that the aortic ring assay can be used to study the angiogenic potential of any physically supporting cell type when under investigation for angiogenic regenerative applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Alpha-MEM</td>
			<td >Gibco</td>
			<td >12571071</td>
			<td >For FTM HUCPVC and BMSC culture media.</td>
		</tr>
		<tr >
			<td >APC-conjugated anti human TRA-1-85</td>
			<td >R&#38;D Systems</td>
			<td >FAB3195A</td>
			<td >Human-specific cell marker for flow cytometry and cell sorting</td>
		</tr>
		<tr >
			<td >Basal membrane extract (BME) (Matrigel)</td>
			<td >Corning</td>
			<td >354234</td>
			<td >For aorta embedding</td>
		</tr>
		<tr >
			<td >Bullet-kit</td>
			<td >Lonza</td>
			<td >CC-3162</td>
			<td >Includes: Gentamicin/Amphotericin-B 
			(GA)human Epidermal 
			Growth Factor (hEGF); Vascular 
			Endothelial Growth Factor (VEGF); R3- 
			Insulin-like Growth Factor-1 (R3-IGF-1); 
			Ascorbic Acid; Hydrocortisone; 
			human Fibroblast Growth Factor-Beta (hFGF-&#946;); Heparin; Fetal Bovine Serum 
			(FBS). Required to prepare EGM</td>
		</tr>
		<tr >
			<td >CellTracker Green CMFDA Dye</td>
			<td >Thermo-fisher</td>
			<td >C2925</td>
			<td >For staining MSCs, green is picked up optimally by MSCs</td>
		</tr>
		<tr >
			<td >CKX53 Culture Microscope</td>
			<td >Olympus</td>
			<td ></td>
			<td >For bright-field imaging of endothelial network development</td>
		</tr>
		<tr >
			<td >Countess automated cell counter</td>
			<td >Invitrogen</td>
			<td >C10227</td>
			<td >Cell counting for MSC culture, flow cytometry and qPCR</td>
		</tr>
		<tr >
			<td >Dispase</td>
			<td >StemCell technologies</td>
			<td >7923</td>
			<td >For dissociating aortic ring-MSC co-cultures (pre-warm at 37 &#176;C)</td>
		</tr>
		<tr >
			<td >Disposable sterile scalpels</td>
			<td >VWR</td>
			<td >21909-654</td>
			<td >For sectioning aorta</td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s phosphate buffered saline</td>
			<td >Sigma-Aldrich</td>
			<td >D8537</td>
			<td >PBS. 1X, Without calcium chloride and magnesium chloride</td>
		</tr>
		<tr >
			<td >Endothelial basal media (EBM)</td>
			<td >Lonza</td>
			<td >CC-3156</td>
			<td >Basal media required for culturing aortic ring assay-MSC co-cultures (warm at 37 &#176;C before use). Required for EGM and EBM-FBS</td>
		</tr>
		<tr >
			<td >Ethanol, 70%, Biotechnology Grade</td>
			<td >VWR</td>
			<td >97064-768</td>
			<td >To sterilize surfaces</td>
		</tr>
		<tr >
			<td >EVOS</td>
			<td >Life Technologies</td>
			<td ></td>
			<td >In-house fluorescent microscope to track MSC migration and integration</td>
		</tr>
		<tr >
			<td >Fetal bovine serum (FBS) (Hyclone)</td>
			<td >GE Healthcare</td>
			<td >SH3039603</td>
			<td >Serum component of cell culture medium</td>
		</tr>
		<tr >
			<td >FITC-conjugated anti-CD31 antibody</td>
			<td >BD</td>
			<td >558068</td>
			<td >Human endothelial marker for flow cytometry</td>
		</tr>
		<tr >
			<td >FITC-conjugated anti-CD146antibody</td>
			<td >BD</td>
			<td >560846</td>
			<td >Human pericyte marker for flow cytometry</td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td >Almedic</td>
			<td >7727-A10-704</td>
			<td >For handing rat tissue. Can use any similar forceps</td>
		</tr>
		<tr >
			<td >Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td >Life Technologies</td>
			<td >14175-094</td>
			<td >1X Without calcium chloride and magnesium chloride</td>
		</tr>
		<tr >
			<td >HERAcell 150i CO2 Incubator</td>
			<td >Thermo Fisher Scientific</td>
			<td >51026410</td>
			<td >For incubating cells</td>
		</tr>
		<tr >
			<td >Human Angiogenesis RT2 profiler PCR array</td>
			<td >Qiagen</td>
			<td >PAHS-024Z</td>
			<td >Human specific and includes primers for 84 genes involved in angiogenesis. Each well is 1 primer reaction</td>
		</tr>
		<tr >
			<td >ImageJ</td>
			<td ></td>
			<td ></td>
			<td >Open source image processing software. Require Angiogenesis analyzer plugin</td>
		</tr>
		<tr >
			<td >LSR II</td>
			<td >BD</td>
			<td ></td>
			<td >UHN SickKids FC Facility. For flow cytometry.</td>
		</tr>
		<tr >
			<td >MoFlo Astrios</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td >UHN SickKids FC Facility. For cell sorting.</td>
		</tr>
		<tr >
			<td >Penicillin/streptomycin</td>
			<td >Gibco</td>
			<td >15140122</td>
			<td >Antibiotic component to buffers and cell culture medium</td>
		</tr>
		<tr >
			<td >RNeasy Mini Kit</td>
			<td >Qiagen</td>
			<td >74104</td>
			<td >For RNA purification. Includes cell lysis buffer</td>
		</tr>
		<tr >
			<td >RT2Easy First Strand Kit</td>
			<td >Qiagen</td>
			<td >330421</td>
			<td >For preparation of cDNA for qPCR</td>
		</tr>
		<tr >
			<td >RT2PreAMP cDNA Synthesis Kit</td>
			<td >Qiagen</td>
			<td >330451</td>
			<td >Pre-amplification of cDNA if low-yield RNA</td>
		</tr>
		<tr >
			<td >Surgical scissors</td>
			<td >Fine Science Tools</td>
			<td >14059-11</td>
			<td >For cutting skin, muscle and aorta</td>
		</tr>
		<tr >
			<td >Sterile gauze</td>
			<td >VWR</td>
			<td >3084</td>
			<td >To dampen and sterilize chest fur</td>
		</tr>
		<tr >
			<td >TrypleE</td>
			<td >Thermo Fisher Scientific</td>
			<td>12605036</td>
			<td >MSC dissociation enzyme pre-warm at 37 &#176;C</td>
		</tr>
		<tr >
			<td >0.2 &#956;m pore filtration unit</td>
			<td >Thermo Fisher Scientific</td>
			<td >566-0020</td>
			<td >To sterilize tissue culture media</td>
		</tr>
		<tr >
			<td >0.25% Trypsin/EDTA</td>
			<td >Gibco</td>
			<td >25200056</td>
			<td >For cell dissociation, pre-warm at 37 &#176;C</td>
		</tr>
		<tr >
			<td >10 cm tissue culture dishes</td>
			<td >Corning</td>
			<td >25382-428</td>
			<td >For cleaning and sectioning aorta and MSC cell culture</td>
		</tr>
		<tr >
			<td >12 well-cell culture plates</td>
			<td >Corning-Sigma Aldrich</td>
			<td >CLS3513</td>
			<td >For setting up aortic ring assay-MSC co-cultures</td>
		</tr>
		<tr >
			<td >15 mL tube</td>
			<td >BD Falcon</td>
			<td >352096</td>
			<td >For general tissue culture procedures</td>
		</tr>
		<tr >
			<td >70 &#956;m cell strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td >To ensure a single cell suspension before flow cytometry or sorting</td>
		</tr>
	</tbody>
</table>

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 schematic work-flow for establishing the aortic ring/MSC co-culture assay is demonstrated in Figure 1. The main steps include: rat aorta isolation, sectioning and embedding of the aortic rings, monitoring the endothelial sprouting and network development, and finally labelling and administering the MSCs. The timeline of the endothelial network analysis outlines the window for the analyses feasible for each period of co-culturing: day 1, 5 &#38;&#160;7. Ad...

Watch this Scientific Journal Video about The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro at JoVE.com

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.

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 ...

The overall goal of this pre-clinical functional assay is to observe and quantitatively evaluate the angiogenic potential of mesenchymal stem cells intended for regenerative therapy. This method can help answer key questions in the field of vascular targeting regenerative medicine, such as the mode of action and the efficiency of cell therapy candidates. The main advantage of this techniques is that it provides key elements of the in vivo environment while providing control and easy assessment characteristics to in vitro assays.The implications of this novel technique extend toward cellular therapy of ischemic diseases, as the field is really missing reliable and easy-to-use functional assays. Demonstrating this procedure will be Farwah Iqbal, a PhD student in my laboratory, as well as Peter Szaraz, a research associate in my laboratory. After isolating a rat aorta and preparing media according to the text protocol, coat 12 well tissue culture plates by first placing the plates on a cold surface such as an ice pack.Using refrigerated pipettes, add 200 microliters per well of freshly thawed basal membrane extract or BME to the wells of the plate. Then, to avoid uneven polymerization of the BME, quickly swirl the BME to ensure even coating. Placing the coated plates into a humidified incubator for 30 minutes.Return the 1, 000 microliter pipette tips back into the minus 20 degrees Celsius freezer. To reduce variability between ring units caused by residual tissue, place the aorta in a 10 centimeter dish with 10 milliliters of fresh HBSS and use two sets of forceps to carefully separate the excess connective and adipose tissue. Use surgical scissors to separate the remaining branches of the intercostal arteries connected to the aorta and remove any residual coagulated blood from the inside of the aorta.Place a ruler or grid under the 10 centimeter dish and use a sterile scalpel and forceps to precisely cut one to two millimeter wide sections of the aorta. To embed the aortic rings in BME after removing the BME coated plates from the incubator, use forceps to carefully pick up individual aortic rings and place each one into the middle of a BME coated well. Use chilled 1, 000 microliter pipette tips to add 300 microliters of BME on top of each aortic ring and evenly distribute the BME around the well.When all of the aortic rings are embedded, return them to the humidified incubator for 30 minutes. Next, following the incubation, add 1, 000 microliters of previously prepared endothelial growth medium or EGM by placing the pipette tip against the wall of the culture well and slowly expelling the solution. Then incubate the tissue for 24 hours.While replacing the medium with EGM containing FBS every 48 hours, use bright-field microscopy to follow the development of the aortic ring endothelial network. To prepare aortic ring assay MSC co-cultures after using bright-field microscopy to image the rat endothelial networks at baseline, seed 10 to the fourth MSCs onto each embedded aortic ring. Maintain at least three wells of aortic rings without MSCs for a control group.Next, visualize the fluorescently labeled MSCs in the aortic ring assay. Following a 24 hour incubation of the aortic ring assay MSC co-cultures, use fluorescence microscopy to visualize MSC homing, elongation, and integration with the endothelial networks. Overlap the fluorescence images of MSCs with bright-field images of endothelial cells to observe the co-localization of both cell types.On days three, five, and seven of the co-cultures with bright-field microscopy, image the aortic ring networks. Use the images to quantify the net effect of MSC co-cultures on endothelial network development. After one week of aortic ring assay MSC co-culture, remove the culture medium and slowly add one milliliter of PBS to each well.Incubate the co-cultures for three minutes and then remove the buffer before repeating the wash two more times. Add 800 microliters of pre-warmed Dispase to each well and incubate the cultures in the humidified incubators for 15 minutes. Following the incubation, pipette the Dispase five to 10 times to break up the VME, and transfer the contents of each well into separate 15 milliliter tubes.Add 800 microliters of fresh pre-warmed Dispase to the same wells and pipette until no residual BME is observed in the culture well. Transfer Dispase cell suspensions to 15 milliliter tubes and use pipette tips to remove the aortic rings floating in the cell suspension. Then add one volume of PBS supplemented with 3%FBS to the Dispase cell suspension to inactivate the Dispase.Spin the cell suspensions at 400 times g for five minutes. Carefully remove the supernatant, leaving three milliliters of cell suspension in the 15 milliliter tube. Add three milliliters of pre-warmed 0.5%trypsin to the cell suspension and vigorously re-suspend the cells.Incubate the trypsinized cell solutions in the humidified incubators for 10 minutes. Following the incubation, add six milliliters of PBS supplemented with 3%FBS, and re-suspend a few times. Then spin the cell suspensions again.Carefully remove all the supernatant, leaving the cell pellet in the tube and use one milliliter of PBS supplemented with 3%FBS to re-suspend the cells. The basal membrane extract can make it challenging to recover the embedded cells. However, Dispase treatment followed by trypsin provides a high yield of viable cells.Using a 70 micron cell strainer, filter the one milliliter cell suspension to remove aggregated cells and residual BME from the cell suspension. Then with a cell counter, count the cells in one milliliter of cell suspension. Carry out flow cytometry and qPCR according to the text protocol.As shown here, three to five days after aortic rings are embedded in ECM, there is a region characterized by high cell proliferation but low structural organization in the vicinity of the aortic tissue. Developed endothelial networks with high structural organization are fully established and predominantly composed of closed loops, even prior to the addition of MSCs. Developing networks are located at the distal areas of the endothelial cultures and are sites of endothelial cell migration and new structure development.After 24 hours in co-cultures, labeled FTM HUCPVCs were found at the periphery of the developing endothelial networks, displayed elongated cell morphologies, and contributed to the further development of endothelial networks. By comparison, BMSC is home to the developing networks while displaying less interaction with endothelial cells. High magnification images at 72 hours showed that FTM HUCPVCs maintained high coverage and stabilization of endothelial networks, while BMSCs and co-cultures, presented with spherical morphologies with limited integration into endothelial networks.Finally, in these images, pre-labeled FTM HUCPVCs are shown adhering with unstained endothelial cells and provide structural support by serving as an axis and attachment surface between nodes. Once mastered, this technique can be done in four hours of hands-on time if it is performed properly. While attempting this procedure, it is important to treat all samples in the same manner and to keep timing stringent.This applies to both the basal membrane extract and the aortic tissue. Following this procedure, other methods like lymphocyte assays can be performed in order to answer additional questions like whether the tested cells changed their mutagenicity in a microvascular environment. After its development, this technique enables researchers in the field of mesenchymal stem cell therapy to explore the angiogenic potential of cell therapy candidates in a multiplex and quantitative way.After watching this video, you should have a good understanding on how to establish the rat aortic ring assay so you can observe and evaluate the behavior of therapeutic cells in developing microvasculature. Don't forget that working with primary animal and human tissue can be hazardous, and precautions such as aseptic technique and personal protective equipment should always be worn while performing this procedure.

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