Name: isolation and profiling of human primary mesenteric arterial endothelial cells at the transcriptome level
Uploaded: 2022-03-14T14:00:00.000+00:00
Duration: 9 min 45 s
Description: Watch this Scientific Journal Video about Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level at JoVE.com

This work was supported by NIH grants R01HL108735, R01HL145170, R01HL106089 (to Z.B.C.); DP1DK126138 and DP1HD087990 (to S.Z.); an Ella Fitzgerald Foundation grant and a Wanek Family Project (to Z.B.C.); and a Human Cell Atlas seed network grant (to Z.B.C. and S.Z.). Research reported in this publication included work performed in the Integrative Genomics Core at City of Hope supported by the National Cancer Institute of the National Institutes of Health under award number P30CA033572. The authors would like to thank Dr. Ismail Al-Abdullah and Dr. Meirigeng Qi of the islet transplantation team at City of Hope for isolation of human tissues, Dr. Dongqiang Yuan at City of Hope for his assistance with scRNA-seq analysis, and Dr. Marc Halushka at the Division of Cardiovascular Pathology, Johns Hopkins University School of Medicine for his invaluable insights into vascular histology.

Human tissue studies were conducted on deidentified specimens obtained from the Southern California Islet Cell Resource Center at City of Hope. The research consents for the use of postmortem human tissues were obtained from the donors&#39; next of kin, and ethical approval for this study was granted by the Institutional Review Board of City of Hope (IRB No. 01046).
1. Physical dissociation (estimated time: 1-2 h)
<ol>
	<li>Place a fresh artery on a 10 cm dish and wash with sterile Dulbecco&#39;s phosphate-buffered saline (D-PBS).</li>
	<li>With sterile forceps and dissection scissors, remove the fat and outer connective tissues until the vessel is clean. Measure the length of the vessel with a ruler placed outside of the dish and take pictures for records (Figure 1A).</li>
	<li>Pre-warm appropriate digestion buffer to 37 &#176;C: for scRNA-seq use cell-dissociation enzyme; for cell culture assays use 1-6 mg/mL of Collagenase D, 3 mg/mL of bacteria-derived protease, 100 mM of HEPES, 120 mM of NaCl, 50 mM of KCl, 5 mM of glucose, with 1 mM of CaCl26,38 (pH 7.0, does not require adjustment) added 5 min prior to use.</li>
	<li>Take the mesenteric artery (typically with a diameter of 6-8 mm) and cut lengthwise with scissors to open the vessel lumen vertically.</li>
	<li>Using needles, attach the vessel onto black wax on all four corners, leaving the intima exposed (Figure 1B).</li>
	<li>Add 1 mL of pre-warmed digestion buffer to intima. Take a sterile scalpel and scrape the lumen of the vessel gently twice. 
	NOTE: The purpose of this procedure is to dissociate the intimal layer, which may need practice. Enough force needs to be exerted to remove endothelium, but not so much that the deeper layers of tissue are also removed, which will reduce the EC representation.</li>
	<li>Transfer the digestion buffer into a 5 mL tube. Add 1 mL of digestion buffer to the intima and pipette up and down carefully to collect the remaining cells and add to the 5 mL tube. Incubate cells at 37 &#176;C, rotating at 150 rpm for 5 min.</li>
	<li>Add 2 mL of M199 medium (or D-PBS if proceeding with scRNA-seq) to the cell suspension to quench the enzymatic reaction. Mix gently and centrifuge at 4 &#176;C, 600 x g for 5 min.</li>
	<li>Remove the supernatant and store the supernatant separately to culture as a control to observe if all cells are captured in the pellet in step 1.8. Resuspend the cell pellet in 1 mL of M199 medium (or D-PBS if proceeding with scRNA-seq).</li>
	<li>Assess the cell viability by mixing 10 &#181;L of trypan blue with 10 &#181;L of cell stock. Observe the cell morphology and count the cells using a hemocytometer. 
	&#8203;NOTE: At this point, cells can be used for protein or RNA quantification or cultured in vitro using EC culture media following standard protocols39. Alternatively, they can be prepared for sequencing as described in the next section.</li>
	<li>For the culture, coat two wells of a 6-well plate by pipetting 500 &#181;L of attachment reagent into wells for 30 min at room temperature. After removal of attachment reagent and washing with sterile D-PBS, dispense the full cell stock into one well, and the supernatant kept as a control into the second well.</li>
</ol>
2. Preparation for scRNA-seq studies (estimated time: 3-4 h)
<ol>
	<li>Centrifuge the cell stock from step 1.11 at 4 &#176;C, 600 x g for 5 min. Remove the supernatant and resuspend the pellet gently with a P1000 pipette and wide-bore tip in 1 mL of 0.04% bovine serum albumin (BSA) in D-PBS. Mix well to ensure a single-cell suspension.</li>
	<li>Pass the solution through a 40 &#181;m strainer to remove cell debris. If debris remains, pass through a second strainer into 4 mL of 0.04% BSA in D-PBS.</li>
	<li>Centrifuge at 4 &#176;C, 600 x g for 5 min. Remove the supernatant and resuspend the pellet in 500 &#181;L of 0.04% BSA in D-PBS using a P1000 pipette with a wide-bore pipette tip. Mix well to ensure a single-cell suspension.</li>
	<li>Assess the cell viability by mixing 10 &#181;L of cell stock with 10 &#181;L of trypan blue. Using a hemocytometer, observe the morphology, determine whether there is a single-cell suspension without clusters, check for tissue debris, and calculate the number of living and dead cells.</li>
	<li>If cells are clustered or debris remains, repeat washing with 0.04% BSA in D-PBS or pass through 40 &#181;m strainer again. 
	NOTE: While this will reduce the yield, it is important that cells exist in a clean single-cell suspension for optimal sequencing.</li>
	<li>The single-cell suspension can be used for scRNA-seq.</li>
</ol>
3. Spatial transcriptome profiling (estimated time: 3-4 h)
<ol>
	<li>Add isopentane (2-methylbutane) to a metal canister and chill in liquid nitrogen (LN2) or dry ice (LN2 is preferred as it will bring the temperature lower than dry ice). Pour optimal cutting temperature compound (OCT) in wells of labeled plastic cryomolds taking care to not create bubbles and remove those that appear. Leave cryomold on dry ice to chill.</li>
	<li>Cut at least two coronal sections, 1 cm in length of the vessel. Using long (12&#34;) forceps, submerge the tissue in isopentane until frozen. Quickly submerge the tissue in OCT at orientation with the lumen visible in the center. Take care to remove any bubbles, especially those next to the tissue.</li>
	<li>Using long (12&#34;) forceps, grasp the cryomolds, and hold them in the metal canister. While the base of the molds should be in the liquid, it should not be too deep to allow isopentane to run over the tissue. Observe the freezing, the OCT will become white progressively from the outside in 1-2 min.</li>
	<li>Store these sections in a sealed container at -80 &#176;C for up to 6 months. 
	NOTE: Maintain samples on dry ice at all times and do not allow more than one freeze-thaw cycle. This tissue can be used for histological analysis, RNA/DNA fluorescence in situ hybridization (FISH), or spatial transcriptomics, which will be described now.</li>
	<li>Set up the cryostat temperature to -20 &#176;C for the chamber and -10 &#176;C for the specimen head. Equilibrate OCT embedded vessel sections, knives, brushes, and slides to -20 &#176;C in the cryostat for approximately 30 min. While equilibrating, clean the machine and all equipment that may touch the sections, including the blade, with 70% ethanol followed by RNase decontamination solution.</li>
	<li>Attach the sample by dispensing a small amount of OCT onto the circular cryostat block and placing the sample on top before it freezes. Place the block into the center of the specimen head and screw the block in place using a tall black handle on the left. Cut away any excess OCT surrounding the vessel.</li>
	<li>Setting the cutting thickness to 10 &#181;m on the cryostat, cut approximately 60 sections and place them in a pre-cooled 1.5 mL tube. These sections will be used to assess the RNA quality. Upon removal from cryostat, immediately add 1 mL of RNA extraction reagent and vortex until the sections are completely dissolved.</li>
	<li>Add 200 &#181;L of chloroform and mix until the solution resembles a strawberry milkshake. Centrifuge at 11,200 x g for 10 min at 4 &#176;C.</li>
	<li>Collect the aqueous phase (clear phase on top of white and pink layers) and add 500 &#181;L of isopropanol to it. Mix by inverting the tubes over 10 times and place at -80 &#176;C for at least 20 min.</li>
	<li>Centrifuge at 11,200 x g for 10 min at 4 &#176;C. Dissolve the purified RNA pellet in 5 &#181;L of RNase-free water. Examine the RNA Integrity Number (RIN). 
	NOTE: Samples with RIN &#8805; 7 are preferred, but RIN &#8805; 6 is acceptable. The number of tissue sections and volume of solution for RNA dissolving may require optimization depending on the system used to ensure the final RNA concentration is within the RIN function range to allow for accurate RIN evaluation.</li>
	<li>Practice cutting and placing sections properly within the fiducial frames using plain glass slides before proceeding to commercially available tissue optimization or gene expression slides. This can be achieved by drawing 6.5 mm x 6.5 mm squares on a plain glass slide, cooling to -20 &#176;C in the cryostat, and placing sections in this capture area.</li>
	<li>Cut 10 &#181;m sections with anti-roll plate in place, flip and carefully flatten by gently touching the section through the surrounding OCT.</li>
	<li>Using RNase-free cryostat brushes place the tissue section within the square, using only the surrounding OCT. Immediately place one finger (in gloves) on the backside of the capture area to melt the section to the slide.</li>
	<li>Once the section has adhered, place the slide onto the cryobar to allow the section to freeze. Care must be taken to avoid tissue folding and tissue overlying the demarcated edges of the fiducial frame. If necessary, cut the tissue into halves or quarters along the lumen using a blade to ensure the vessel section fits within the 6.5 mm x 6.5 mm frame.</li>
	<li>Cut 10 &#181;m sections of tissue as per 3.12-3.14 onto tissue optimization or gene expression slides. Transfer the slide to a slide mailer placed on dry ice. Store slides at -80 &#176;C for up to 4 weeks before proceeding with published spatial protocols33,34.</li>
</ol>
4. Sequencing data analysis (estimated time: up to 1 week depending on familiarity with software)
NOTE: For spatial transcriptomic analysis only, skip to step 4.8. scRNA-seq data is processed using the standardized pipeline aligned to human hg38 reference transcriptome. The R package Seurat (v3.2.2) is used to analyze scRNA-seq data following published guidelines40.
<ol>
	<li>Filter using well-established quality control metrics: rare cells with very high numbers of genes (potentially multiplets) and cells with high mitochondrial percentages (low-quality or dying cells often present mitochondrial contamination) are removed.</li>
	<li>Normalize data using &#34;sctransform&#34;, a method to improve sample integration compared to log-normalization. These normalized data are used for dimensionality reduction and clustering, while log-normalized expression levels are used for analysis based on gene expression levels, such as cell-type classification41.</li>
	<li>Select marker genes per cell type, for example, platelet endothelial cell adhesion molecule-1 (PECAM1) and von Willebrand factor (VWF) for ECs, lumican (LUM) and procollagen C-Endopeptidase Enhancer (PCOLCE) for fibroblasts, B-cell translocation gene 1 (BTG1) and CD52 for macrophages, C1QA and C1QB for monocytes, and myosin heavy chain 11 (MYH11) and transgelin (TAGLN) for vascular smooth muscle cells36.</li>
	<li>Compute the average expression level across single cells between each pair of markers in order to have a single marker with an average expression level associated with each cell type42.</li>
	<li>Apply a Gaussian Mixture Model (GMM) with two components to the expression data of each marker across single cells in order to separate cells into two sets, namely, highly expressing the marker and lowly expressing the marker. In this way, for each marker, each single cell is assigned to one of the two components42.</li>
	<li>Use statistical enrichment for the set of marker genes, a Fisher&#39;s exact test, to assign a cell type to each cluster. By doing so, a set of p-values are obtained per cluster, each corresponding to a cell type. The cell type with the lowest p-value is assigned to that cluster.</li>
	<li>Process the spatial transcriptomic data using the Space Ranger pipelines resulting in spatial de-barcoding and generation of quality control (QC) metrics, including total aligned reads to hg38 human transcriptome, the median number of Unique Molecular Identifier (UMI) or genes per spot35. 
	NOTE: The R package Seurat (v3.2.2) is used to analyze the Space Ranger-processed data following published guidelines40.</li>
	<li>Normalize data using &#34;sctransform&#34;, a method which is demonstrated to improve the downstream analysis compared to log-normalization given the heterogeneity of the tissue and the very high variance in counts across the spots.</li>
</ol>

S.Z. is a founder and board member of Genemo, Inc.

The presented workflow details a set of techniques to profile ECs from a single piece of human artery with single-cell and spatial resolution. There are several critical steps and limiting factors in the protocol. One key to transcriptome profiling is the freshness of the tissue and RNA integrity. It is important to maintain tissues on ice as much as possible prior to processing to minimize RNA degradation. Typically, the post-mortem tissues are processed between 8-14 h after the time of death. However, beginning the iso...

Lining the lumen of blood vessels, endothelial cells (ECs) are crucial regulators of vascular tone and tissue perfusion. ECs are remarkable in their ability to react to the extracellular environment and adapt to changes in the dynamics and composition of blood flow. These dynamic responses are mediated through a network of intracellular signaling events, including transcriptional and post-transcriptional modulations with spatio-temporal resolution. The dysregulation of these responses is implicated in many pathologies, including but not limited to cardiovascular disease, diabetes, and cancer1,2.
A large proportion of studies make use of cell lines or animal models to interrogate EC transcriptome. The former is a useful tool, given the relative ease of use and inexpensiveness. However, serial culturing can introduce phenotypic alterations to ECs, such as fibroblastic features and a lack of polarization, disconnecting them from their in vivo state3. The primary cells, e.g., human umbilical vein EC (HUVEC) have been a popular choice since the 1980s but are derived from a developmental vascular bed that does not exist in adults, thus unlikely to fully represent mature ECs. Animals, especially mouse models, better represent the physiological or pathophysiological environment of ECs and allow interrogation of transcriptomes as a result of genetic perturbation. Murine ECs can be isolated from various tissues, including the aorta, lungs, and adipose tissues using enzyme-based procedures4,5,6,7. However, the isolated cells cannot be used for multiple passages unless transformed6 and are often limited in numbers, which requires pooling from multiple animals5,8,9.
The advent of new technologies exploring the vessel architecture at a transcriptomic level, particularly with single-cell resolution, has enabled a new era of endothelial biology by revealing novel functions and properties of ECs5, 10,11,12,13,14. A rich resource built by Tabula Muris investigators collected single-cell transcriptomic profiles of 100,000 cells including ECs across 20 different murine organs15, which revealed both common EC marker genes and unique transcriptomic signatures with inter-and intra-tissue differences5,13. Nevertheless, there are clear differences between mouse and human in genome, epigenome, and transcriptome, especially in the non-coding regions16,17,18. These aforementioned drawbacks emphasize the importance of analysis of ECs using human samples in order to gain a faithful profile of ECs in their native state in health and disease.
Most EC isolation methods rely on physical dissociation through homogenization, finely cutting, and mincing the tissue before incubation with proteolytic enzymes for differing times. The enzymes and conditions also vary considerably between tissue types, from trypsin to collagenase, used alone or in combination19,20,21. Further antibody-based enrichment or purification are often included to increase the purity of ECs. Typically, antibodies against EC membrane markers, e.g., CD144 and CD31 are conjugated to magnetic beads and added to the cell suspension22,23. Such a strategy can be generally adapted for EC isolation from multiple human and mouse tissues, including the techniques introduced in this protocol.
In their native state, ECs interact with multiple cell types and may exist in vascular niches where cell proximity is crucial for function. While single-cell and single-nuclear RNA-sequencing (scRNA and snRNA-seq) studies have been paramount to the recent breakthroughs in describing EC heterogeneity, the dissociation process disrupts tissue context and cell-cell contact, which are also important to understand EC biology. Developed in 2012 and named Method of the Year in 202024, spatial transcriptome profiling has been utilized to profile global gene expression while retaining the spatial features in various tissues including brain25, tumor26, and adipose tissue27. The technologies can be targeted, using specialized probes specific for particular RNA sequences attached to affinity reagents or fluorescent tags, thus detecting select genes at subcellular resolution28,29,30,31. They can also be untargeted32,33, typically using spatially barcoded oligonucleotides to capture RNA, which together get converted to cDNA for subsequent seq library preparation and hence has the advantage of deducing whole tissue gene expression in an unbiased manner. However, spatial resolution is not currently achieved at a single cellular level with commercially available technologies. This can be overcome to some extent with data integration with scRNA-seq data, ultimately allowing the mapping of single-cell transcriptome in a complex tissue context while retaining its original spatial information34.
Herein, a workflow is described to profile EC transcriptome using human superior mesenteric artery, a peripheral artery that has been used to study vasodilation, vascular remodeling, oxidative stress, and inflammation35,36,37. Two techniques are described: 1) to isolate and enrich ECs from the intima of blood vessels combining mechanical dissociation and enzymatic digestion suitable for single-cell transcriptome sequencing or subsequent in vitro culture; 2) to prepare arterial sections for spatial transcriptome profiling (Figure 1). These two techniques can be performed independently or complementarily to profile ECs and their surrounding cells. Furthermore, this workflow can be adapted for use on any medium or large artery.

<table><tbody><tr><td>1.5 mL micro-centrifuge tube</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>10 cm dish</td><td>Genesee Scientific</td><td>25-202</td><td></td></tr><tr><td>23G needles</td><td>BD</td><td>305145</td><td></td></tr><tr><td>2-methylbutane</td><td>Thermo Fisher</td><td>AC327270010</td><td></td></tr><tr><td>40 &amp;micro;m strainer</td><td>Fisher</td><td>14100150</td><td></td></tr><tr><td>4200 TapeStation System</td><td>Agilent Technologies</td><td>G2991BA</td><td></td></tr><tr><td>5 mL tube</td><td>Thermo Fisher</td><td>14282300</td><td></td></tr><tr><td>6-well plate</td><td>Greiner Bio-One</td><td>07-000-208</td><td></td></tr><tr><td>Attachment factor</td><td>Cell Applications</td><td>123-500</td><td>Attachment reagent in the protocol</td></tr><tr><td>Black wax</td><td></td><td></td><td>Any commercial black wax can be used</td></tr><tr><td>Bovine serum albumin heat shock treated</td><td>Fisher</td><td>BP1600-100</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>BP510</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Chloroform</td><td>Fisher</td><td>C607</td><td></td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088866001</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td></td><td></td></tr><tr><td>Cryostat brushes</td><td></td><td></td><td></td></tr><tr><td>D-Glucose</td><td>Fisher</td><td>D16-1</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Fisher</td><td>MT25950CQC</td><td></td></tr><tr><td>Dispase II</td><td>Roche</td><td>4942078001</td><td>Bacteria-derived protease in the protocol</td></tr><tr><td>Disposable Safety Scalpels</td><td>Myco Instrumentation</td><td>6008TR-10</td><td></td></tr><tr><td>D-PBS</td><td>Thermo Fisher</td><td>14080055</td><td></td></tr><tr><td>Ethanol</td><td>Fisher</td><td>BP2818-4</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Fisher</td><td>10437028</td><td></td></tr><tr><td>Hemocytometer</td><td>Fisher</td><td>267110</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375-100g</td><td></td></tr><tr><td>High sensitivity D1000 sample buffer</td><td>Agilent Technologies</td><td>5067-5603</td><td></td></tr><tr><td>High sensitivity D1000 screen tape</td><td>Agilent Technologies</td><td>5067-5584</td><td></td></tr><tr><td>Incubator</td><td></td><td></td><td>Kept at 37 &amp;deg;C 5% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Isopropanol</td><td>Fisher</td><td>BP26324</td><td></td></tr><tr><td>KCl</td><td>Fisher</td><td>P217-3</td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Medium 199</td><td>Sigma Aldrich</td><td>M2520-10X</td><td></td></tr><tr><td>Metal cannister</td><td></td><td></td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td></td><td>To assess cell morphology</td></tr><tr><td>Microvascular endothelial culture medium</td><td>Cell Applications</td><td>111-500</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>S271-1</td><td></td></tr><tr><td>New Brunswick Innova 44/44R Orbital shaker</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature compound</td><td>Fisher</td><td>4585</td><td></td></tr><tr><td>Plastic cryomolds</td><td>Fisher</td><td>22363553</td><td></td></tr><tr><td>RNA screen tape</td><td>Agilent Technologies</td><td>5067-5576</td><td></td></tr><tr><td>RNA screen Tape sample buffer</td><td>Agilent Technologies</td><td>5067-5577</td><td></td></tr><tr><td>RNase ZAP</td><td>Thermo Fisher</td><td>AM9780</td><td></td></tr><tr><td>RNase-free water</td><td>Takara</td><td>RR036B</td><td>RNase-free water (2) in kit</td></tr><tr><td>Sterile 12&quot; long forceps</td><td>F.S.T</td><td>91100-16</td><td></td></tr><tr><td>Sterile fine forceps</td><td>F.S.T</td><td>11050-10</td><td></td></tr><tr><td>Sterile fine scissors</td><td>F.S.T</td><td>14061-11</td><td></td></tr><tr><td>Superfrost PLUS Gold Slides</td><td>Fisher</td><td>1518848</td><td></td></tr><tr><td>TRIzol reagent</td><td>Fisher</td><td>15596018</td><td></td></tr><tr><td>Trypan Blue</td><td>Corning</td><td>MT25900CI</td><td></td></tr><tr><td>TrypLE Express Enzyme (1X) phenol red</td><td>Thermo Fisher</td><td>12605010</td><td>Cell-dissociation enzyme in the protocol</td></tr><tr><td>Visium Accessory Kit</td><td>10X Genomics</td><td>PN-1000215</td><td></td></tr><tr><td>Visium Gateway Package, 2rxns</td><td>10X Genomics</td><td>PN-1000316</td><td></td></tr><tr><td>Visium Spatial Gene Expression Slide &amp;amp; Reagent Kit, 4 rxns</td><td>10X Genomics</td><td>PN-1000184</td><td></td></tr></tbody></table>

isolation and profiling of human primary mesenteric arterial endothelial cells at the transcriptome level

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the transcriptome and epigenome of ECs is paramount to understanding their roles in development, health, and disease, but is limited in the availability of isolated primary cells. Recent technologies have enabled the high-throughput profiling of EC transcriptome and epigenome, leading to the identification of previously unknown EC cell subpopulations and developmental trajectories. While EC cultures are a useful tool in the exploration of EC function and dysfunction, the culture conditions and multiple passages can introduce external variables that alter the properties of native EC, including morphology, epigenetic state, and gene expression program. To overcome this limitation, the present paper demonstrates a method of isolating human primary ECs from donor mesenteric arteries aiming to capture their native state. ECs in the intimal layer are dissociated mechanically and biochemically with the use of particular enzymes. The resultant cells can be directly used for bulk RNA or single-cell RNA-sequencing or plated for culture. In addition, a workflow is described for the preparation of human arterial tissue for spatial transcriptomics, specifically for a commercially available platform, although this method is also suitable for other spatial transcriptome profiling techniques. This methodology can be applied to different vessels collected from a variety of donors in health or disease states to gain insights into EC transcriptional and epigenetic regulation, a pivotal aspect of endothelial cell biology.

The analysis of ECs from mesenteric artery using a combination of mechanical and enzymatical dissociation or cryopreservation for use in various downstream assays is depicted here (Figure 1). ECs can be profiled in mesenteric arteries using the following steps: A) mechanical dissociation from the intima coupled with collagenase digestion to culture cells; B) generation of single-cell suspension for scRNA-seq; or C) cross-sections of the artery can be embedded in OCT to be cryosectioned to pr...

Watch this Scientific Journal Video about Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level at JoVE.com

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the ...

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Medicine

2.9K Views.  City of Hope. The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.

Video: Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Cancer Research

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Developmental Biology

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Neuroscience

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Biology

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Immunology and Infection

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...