We would like to thank Andrea Portbury for critical reading of the manuscript, the Microscope Service Laboratory of UNC for assistance with the time-lapse imaging, and the NIH (grant # R01HL061656) for funding support.

1. Prepare Antibody-conjugated Beads
<ol>
 <li>One day prior to dissection, combine the antibody of choice with the appropriate Dynabeads (e.g. rat IgG Dynabeads for an antibody raised in rat, 4 x 108 beads/ml) per the manufacturer's instructions. For the BD rat anti-CD31 antibody (0.5 &mu;g/&mu;l, used to isolate endothelial cells), 3 &mu;l antibody is added for every 25 &mu;l beads, for a final concentration of 1.5 &mu;g anti-CD31 antibody and 1 x 107 beads/25 &mu;l buffer.</li>
 <li>Incubate beads and antibody overnight at 4 &deg;C.</li>
</ol>
2. Preparing the Collagen Plate
<ol>
 <li>Isolated cells form tubules more quickly when grown on a collagen gel as opposed to merely a collagen-coated plate. Therefore, collagen gels are made the night before the dissection, as described in 6. On ice in a tissue culture hood, combine the following reagents (enough for 19 wells): 206.4 &mu;l sterile H2O, 140.4 &mu;l collagen type I (4.08 mg/ml), 38.4 &mu;l 10x M199, and 1.15 &mu;l 5 M NaOH.</li>
 <li>Pipette 20 &mu;l of this collagen gel into the wells of a 384-well tissue culture plate. Place the plate in a 37 &deg;C tissue culture incubator for 30 min.</li>
 <li>Add 80 &mu;l DMEM to each well and incubate for 15 min at 37 &deg;C. Remove the culture medium and repeat twice more.</li>
 <li>After the final rinse, add 80 &mu;l 10% FBS/DMEM to each well and incubate overnight at 37 &deg;C. Remove the culture medium prior to adding the isolated cells.</li>
</ol>
3. Excise and Digest the Heart
<ol>
 <li>Euthanize a timed-pregnant mouse at the desired embryonic day using an approved euthanasia technique. All experiments were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill.</li>
 <li>With the mouse in a supine position, liberally spray the ventral side of the female with 70% ethanol prior to dissection. Using forceps to lift the lower abdominal skin and muscle away from the internal organs, cut open the female's lower abdominal cavity to visualize the internal organs.</li>
 <li>Holding the cervix with forceps, carefully cut caudal to the forceps and begin lifting the uterine horn from the abdominal cavity. While lifting the uterine horn, cut away any connective tissue that holds the horn in place. To finish excising the uterine horn, cut the junction of the uterine horn with the oviduct to free the uterine horn.</li>
 <li>Place the uterine horn in a Petri dish containing cold 1x phosphate-buffered saline (PBS) and rinse as needed. Cut open the uterine horn via the midline to expose the attached embryonic sacs.</li>
 <li>Cut open an embryonic sac at the junction of the placenta and the embryo to avoid accidentally cutting the embryo and retrieve an embryo. Place the embryo in a second Petri dish with cold PBS. Return the Petri dish with the uterine horn to ice.</li>
 <li>Decapitate the embryo to improve access to the chest wall. If genotyping is necessary, cut the tail to remove it and save in an Eppendorf tube placed on ice.</li>
 <li>With the embryo on its back, cut open the chest wall to visualize the heart and lungs. To avoid cutting the heart, make a vertical cut along the side of the rib cage, near a forelimb, followed by a horizontal cut across the bottom of the ribs; then, gently pull the chest wall up and out of the way. Up through approximately embryonic day 13.5, the chest wall is transparent, aiding in visualization.</li>
 <li>Carefully lift the heart using forceps and cut the vessels below. Then, cut above the great vessels to free the heart. If the pulmonary vessels are not cut, the lungs may be removed with the heart. Thus, remove extraneous tissues, like the lungs, if necessary.</li>
 <li>Separate and discard the atria and great vessels from the ventricles. Using scissors, mince the ventricles into small pieces (approximately 1 mm3) and place in approximately 500 &mu;l collagenase (1 mg/ml in sterile PBS). Keep samples on ice until all ventricles have been minced and placed in collagenase.</li>
 <li>Repeat the previous steps until the ventricles have been excised from all embryos, collecting each heart in a separate Eppendorf tube. If all embryos have the same genotype, hearts may be pooled in a single Eppendorf tube. The number of samples processed at a single time may be limited based on the magnet used for isolation; the DynaMag (123-21D) used here holds a maximum of 16 Eppendorf tubes.</li>
 <li>Place the minced ventricles at 37 &deg;C with rocking for 45 min. Every 15 min, gently remove the samples and pipette up and down to mechanically dissociate the cells.</li>
</ol>
4. Isolating the Endothelial Cells
<ol>
 <li>Pass the ventricle-collagenase solution through a 70 &mu;m filter to remove remaining clumps of cells.</li>
 <li>Centrifuge the cells at 400 x g for 10 min. Discard the supernatant, replace with approximately 200 &mu;l 10% FBS in DMEM, and pipette up and down to dissociate the cells.</li>
 <li>Repeat the previous step once. Centrifuge the cells at 400 x g for 10 min after the second 10% FBS/DMEM wash. Resuspend the cells in 50 &mu;l 10% FBS/DMEM.</li>
 <li>During the final centrifugation step, prepare the beads.
 <ol>
 <li>Place the Eppendorf tube containing the antibody and beads on a magnet for 1 min.</li>
 <li>With the tube in place on the magnet, remove the supernatant. Remove the tube from the magnet and add approximately 100 &mu;l 10% FBS/DMEM.</li>
 <li>Place the tube on the magnet for 1 min. Remove the supernatant, and wash again with 10% FBS/DMEM. Repeat for a total of 3 washes.</li>
 <li>After the final wash, resuspend the antibody-conjugated beads in 10% FBS/DMEM (5 &mu;l per sample).</li>
 </ol>
 </li>
 <li>To each cell sample, add 5 &mu;l of the antibody-conjugated beads. Place the bead-cell suspensions at 4 &deg;C with rocking for 30 min.</li>
 <li>In a laminar flow hood, place the Eppendorf tubes containing cells and antibody-conjugated beads on the magnet for 1 min. Remove the supernatant, remove the tubes from the magnet, and wash with approximately 100 &mu;l 10% FBS/DMEM. Repeat for 5 washes.</li>
 <li>After the last wash, place the tubes on the magnet for 1 min, remove the supernatant, and remove the tubes from the magnet. Add approximately 100 &mu;l DMEM.</li>
 <li>Place the tubes on the magnet for 1 min, remove the supernatant, and remove the tubes from the magnet. Add 100-200 &mu;l prewarmed 0.25% trypsin-EDTA. Incubate the samples at 37 &deg;C and 5% CO2 for 5 min.</li>
 <li>Place the tubes on the magnet for 2 min. Carefully transfer the cell-containing supernatant to fresh Eppendorf tubes. Centrifuge for 5 min at 400 x g.</li>
 <li>Remove the supernatant. Resuspend the cells in 80-100 &mu;l growth medium and plate in a 96- or 384-well treated culture dish, depending on the expected yield (approximately 450-600 cells from the ventricles of E14.5-E16.5 embryos). For endothelial cells, use endothelial growth culture medium (defined below).</li>
</ol>

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Hemost. 36, 227-235 (2010).</li><li>Dong, Q. G., Bernasconi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+strategy+for+isolation+of+endothelial+cells+from+murine+tissues.+Characterization+of+two+endothelial+cell+lines+from+the+murine+lung+and+subcutaneous+sponge+implants.">A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 17, 1599-1604 (1997).</li><li>van Beijnum, J. R., Rousch, M., Castermans, K., vander Linden, E., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+lung+endothelial+cells.">Isolation of murine lung endothelial cells.</a> American Journal of Physiology: Lung Cellular and Molecular Physiology. 296, L1095-L1103 (2009).</li><li>Frauli, M., Ludwig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+fibroblast+proliferation+in+a+culture+of+human+endometrial+stromal+cells+using+a+medium+containing+D-valine.">Inhibition of fibroblast proliferation in a culture of human endometrial stromal cells using a medium containing D-valine.</a> Archives of Gynecology and Obstetrics. , 241-96 (1987).</li><li>Lazzaro, V. A., Walker, R. 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The authors declare that they have no competing financial interests.

Working with primary embryonic cells allows novel experiments to address in vitro critical steps of development. However, the isolation procedure is not trivial. Critical steps for isolating any type of cells include ensuring that the cells are well separated upon collagenase digestion and then that they are well suspended during the wash steps. Mechanical dissection by pipetting greatly helps separate the cells during the collagenase step, and the filtering step removes clumps of cells. These steps impro...

The advent of immortalized cell lines has revolutionized basic cell biology 1. The currently available cell lines are derived from a wide range of organs and encompass all the major cell types. However, established cell lines have some limitations. The process of immortalization obviously changes the behavior of the cells, specifically with respect to life span and proliferation, but can also affect the expression of unexpected proteins, such as cytoskeletal proteins 2. In addition, although many different cell lines are available, there is significant diversity among even a single cell type within an entire organism. Endothelial cells in particular show diverse behaviors based on whether they line arteries or veins and what kind of flow is present in the vessel 3. Even more pressing from a developmental perspective, however, is that the vast majority of the available cell lines are derived from adult tissue (see, e.g. the collection available via ATCC). These adult cell lines likely do not recapitulate the dynamic nature of their embryonic precursors. The rapidly changing spatiotemporal gene expression patterns observed during development also suggest that an endothelial cell from one organ at a given developmental stage may not behave the same way as an endothelial cell from that same organ at a different developmental stage.
As an alternative to using commercially available immortalized cell lines, primary cells can be isolated from the specific tissue of interest. Among the advantages of this technique, these primary cells can be isolated from a specific organ or even part of an organ at any specific developmental stage. Further, these primary cells can be isolated from the wide variety of available transgenic animals, allowing the in vitro study of gene knockout and knock-in while avoiding other problems such as transfection efficiency. Not surprisingly, many techniques have been published detailing how to isolate specific cell types 4,5. In general, these techniques involve collecting the region of interest, dissociating the cells, tagging the specific cell type of interest, and isolating those cells for further analysis.
To study an early embryonic population of coronary endothelial cells, we scaled down a previously published technique4 for use with a smaller organ. With this scaled-down procedure, we can isolate the coronary endothelial cells from the embryonic heart at specific embryonic stages. These cells can then be used in traditional endothelial assays, such as migration analyses. Until early embryonic cell lines become more prevalent, working with the primary cells is an invaluable technique.

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>Timed-pregnant mice</td><td></td><td></td><td>To be dissected at the embryonic stage of interest</td></tr><tr><td>Anti-mouse CD31</td><td>BD Bioscience</td><td>553370</td><td></td></tr><tr><td>Rat IgG Dynabeads</td><td>Invitrogen</td><td>110-35</td><td></td></tr><tr><td>Collagen</td><td>BD Bioscience</td><td>354236</td><td></td></tr><tr><td>PBS (1x)</td><td></td><td></td><td></td></tr><tr><td>Collagenase, type I</td><td>Worthington Biochemical</td><td>LS004196</td><td></td></tr><tr><td>DMEM</td><td>Cellgro</td><td></td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F2442</td><td></td></tr><tr><td>Endothelial cell growth medium</td><td>(made in lab as described in notes)</td><td></td><td>DMEM containing 20% FBS, 5 &amp;mu;M &amp;#946;-mercapt&amp;#8211;thanol, 50 &amp;mu;g/ml ECG, and 1x penicillin/streptomycin</td></tr><tr><td>&amp;#946;-mercapt&amp;#8211;thanol</td><td>Sigma-Aldrich</td><td>M6250</td><td></td></tr><tr><td>ECGS</td><td>Biomedical Technologies, Inc.</td><td>BT-203</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25300</td><td></td></tr><tr><td>384-well culture plate</td><td>Greiner</td><td>T-3037-6</td><td>Plate was prepared with a thin collagen gel, as described in &lt;sup&gt;6&lt;/sup&gt;; working surface area 10 mm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Collagen type I</td><td>BD</td><td>354236</td><td>Use at a final concentration of 1.5 mg/ml</td></tr><tr><td>M199, 10x</td><td>Invitrogen</td><td>11825-015</td><td></td></tr><tr><td>Syto-16</td><td>Invitrogen</td><td>S7578</td><td>Used as directed in &lt;sup&gt;13&lt;/sup&gt; </td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Stereoscopic microscope</td><td>Nikon</td><td>SMZ645</td><td></td></tr><tr><td>Cell culture incubator</td><td>Thermo</td><td>3110</td><td></td></tr><tr><td>DynaMag</td><td>Invitrogen</td><td>123-21D</td><td></td></tr><tr><td>Table-top centrifuge</td><td>Thermo</td><td>75002430</td><td></td></tr></tbody></table>

isolation of embryonic ventricular endothelial cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Using an endothelial-specific antibody that recognizes CD31, endothelial cells were isolated from the ventricles of embryonic (E) 13.5 (Figure 1A) and 18.5 (Figures 1B-1D) embryos. When grown on an untreated culture dish, these cells remain rounded and would form a cobblestone pattern if near confluent (Figure 1A). Dilute collagen has also been used to coat the wells, and while the endothelial cells will adhere to it (Figure 1B), they form fewer chains t...

Watch this Scientific Journal Video about Isolation of Embryonic Ventricular Endothelial Cells at JoVE.com

Isolation of Embryonic Ventricular Endothelial Cells

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

isolation-of-embryonic-ventricular-endothelial-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.3K Views.  University of North Carolina at Chapel Hill. Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Video: Isolation of Embryonic Ventricular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Developmental Biology

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Neuroscience

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

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 ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...