This work was supported by the grant from the National Institutes of Health (HL115578 to A. Makino).

The research on mice was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Arizona and was performed according to the National Institute of Health (NIH) guidelines.
NOTE: Sections 1, 2 and 3 should be carried out the day before the experiment as setup.
1. Preparing Solutions
<ol>
	<li>CD31 buffer (50 ml)
	<ol>
		<li>Add 50 ml 1&#215; Phosphate Buffer Saline (PBS) to a 50 ml tube. Weigh 0.05 g Bovine Serum Albumin (BSA) and 0.037 g Ethylenediaminetetraacetic acid (EDTA) and add to the&#160;50 ml tube and put the tube to rotate for about 1 h until powder dissolves. Adjust pH to 7.4, filter through a 0.2 &#181;m filter into a new 50 ml tube, and store at 4 &#730;C until use.</li>
	</ol>
	</li>
	<li>Hank&#39;s Balanced Salt Solution (HBSS) with HEPES (500 ml)
	<ol>
		<li>Add 1.1915 g of&#160;HEPES to make 10 mM HEPES&#160;in&#160;HBSS solution and mix well. Measure the pH and adjust to 7.4. Filter and store at 4 &#730;C.</li>
	</ol>
	</li>
	<li>1x Kreb&#39;s solution (1 L)
	<ol>
		<li>Add 100 ml of 10&#215; Kreb&#39;s solution [Table 1] to double distilled water in a 1 L flask. Weigh 2.1 g Sodium Bicarbonate and 2 g D-Glucose and add to the flask. Mix and adjust the total volume to 1 L.</li>
		<li>Bubble solution with 95% O2/5% CO2 gas for 30 min and filter solution inside the hood. Store at 4 &#730;C and keep on ice during experiment.</li>
	</ol>
	</li>
</ol>
2. Preparing the Magnetic Beads
<ol>
	<li>Add 1 ml CD31 buffer into each 1.5 ml tube (one tube per sample). Mix the sheep anti-rat IgG beads and add the appropriate amount to each sample [Table 1]. Shake the tubes vigorously 30 times and place them in the magnetic plate for 1 min&#160;of shaking. Open the tubes while on the plate and dump the solutions into a bucket.</li>
	<li>Add 1 ml CD31 buffer into each tube. Add 2 &#181;l Rat anti-mouse CD31&#160;antibody to each tube and rotate tubes O/N at 4 &#186;C.</li>
</ol>
3. Autoclaving&#160;
<ol>
	<li>Autoclave all surgery tools and pipette tips. Cut the ends of pipette tips so that there are tips with 4 different diameters, from widest to narrowest. Approximate diameters for the tips are 3 mm, 2.5 mm, 2 mm, and 1.5 mm respectively. Cut the tips and autoclave the day before with 30 min of sterilization at a temperature of 121 &#186;C. 
	NOTE: Sections 4 - 11 should be&#160;carried out on the day of the experiment.</li>
</ol>
4. Preparing Solutions
<ol>
	<li>Prepare washing buffer containing 2% Iron-Fortified Calf Serum (IFCS) in M199. For imaging, prepare&#160;30&#160;ml/mouse.</li>
	<li>Prepare enzyme solution (10 ml/sample).&#160; Weigh 0.6 unit/ml of Dispase and 1 mg/ml of Collagenase Type II into a 50 ml tube. Add M199 and rotate the tube at 4 &#186;C for 15 min. &#160;Filter through 0.2 &#181;m filter into a new 50 ml tube inside the hood and keep at 4 &#730;C until use.</li>
	<li>Prepare HBSS/HEPES with Heparin.&#160; Add 10 ml of HBSS with 10 mM HEPES in 15 ml tube. Add 100 &#956;l of Heparin to the tube and keep at 4 &#730;C until use.</li>
</ol>
5. Washing the Beads
<ol>
	<li>Take out the tubes with beads from the rotator at 4 &#186;C and bring to the hood. Place the tubes in the magnetic plate and shake for 1 min. Dump the solution and add 1 ml CD31 buffer into each tube.</li>
	<li>Take the tubes out of the magnetic plate and shake vigorously 30 times. Put the tubes back in the magnetic plate and repeat for a total of 3 washes. After completion, put the tubes back to 4&#730;C and keep rotating until use.</li>
</ol>
6. Dissection
<ol>
	<li>Inject the mice intraperitoneally with 0.1 ml of Heparin to prevent blood coagulation. Wait 10 mins, then anesthetize the mice using intraperitoneal injection of 0.01 ml of Pentobarbital. Check if the mouse is feeling pain by pinching the foot.</li>
	<li>Place the mouse on a polystyrene board with head up and hold the arms with pins. Use forceps and tough-cut-scissors to cut the skin at the upper thoracic area up to the throat.</li>
	<li>Make a cut in the center of the rib cage just above the diaphragm. Cut the bone sideways and upwards toward the throat to make a triangle exposing the heart and lungs. Cut the aorta just above the diaphragm.</li>
	<li>Hold the thymus with forceps without pulling and use scissors to cut any connective tissue carefully to remove lungs and heart together. Place tissues in a beaker with 1&#215; Kreb&#39;s, shake with forceps and transfer to 6 cm dish with 1&#215; Kreb&#39;s.</li>
	<li>Remove the lung lobes using scissors and forceps. Insert a 20 G sterile catheter into the aorta. Flush the blood in the coronary circulatory system with HBSS/Heparin. Make a small cut in the ventricle, shake the heart and transfer to a sample tube of M199. Keep tubes on ice.</li>
</ol>
7. Tissue Digestion
<ol>
	<li>Transfer the heart to a 6 cm dish filled with 10 ml of enzyme solution and mince it into small pieces using scissors. Place the dishes in the 37 &#186;C incubator for 15 min. Agitate digested materials by pipetting up and down 30 times through a tip with a cut end to allow the tissue to pass through easier.</li>
	<li>Put it back in the 37 &#186;C incubator for another 15 min. Repeat agitation three times, each time using pipette tips from biggest openings to narrowest&#160;and 15 min incubation between each.</li>
	<li>After last 15 min incubation, take samples out and into the hood. Pipet solution up and down 30 times using pipet tip with the narrowest opening and pass through a 40 &#181;m cell strainer into a new 50 ml tube.</li>
	<li>Wash the dish with 10 ml washing buffer using a&#160;10 ml pipette and transfer onto cell strainer. Put the 50 ml tubes in the centrifuge and spin at 400 g for 10 min at RT.</li>
</ol>
8. Washing and Conjugation with Beads
<ol>
	<li>Remove supernatant without disrupting the pellet. Wash cells with 5 ml washing buffer twice.</li>
	<li>Get one of the previously prepared 1.5 ml tubes with beads and work on one tube at a time. Put one 1.5 ml tube in the magnetic plate, shake 3 times and open it.</li>
	<li>After last centrifuge, remove as much supernatant as possible without disrupting the pellet. Disassociate the cell clumps by flicking vigorously.</li>
	<li>Add 1 ml washing buffer to the 50 ml tube and mix the cells with a 1 ml pipette. Dump the solution from the 1.5 ml tube and add 1 ml of cell suspension from the 50 ml tube to the beads. Flick the tubes 30 times then rotate at 4 &#186;C for 30 min.</li>
</ol>
9. Preparing for Cell Culture/Imaging
<ol>
	<li>Prepare chambers for plating cells. Add gelatin solution (5% w/v, 300 &#181;l) to the chamber well to coat the surface and incubate for 30 min at 37 &#186;C. After incubation, remove gelatin and dry the surface.</li>
	<li>Take out the tubes with beads/cells after 30 min of rotation and put in the magnetic plate inside the hood. Shake for 2 min, dump and add 1 ml washing buffer.</li>
	<li>Take tubes out of the plate, shake vigorously 30 times then flick 30 times. Put them back in magnetic plate and shake for 1 min. Repeat 4 times for a total of 5 washes, but with 1 min shaking on the plate.</li>
	<li>After the last wash, take out one tube and work on each tube one at a time. Shake 30 times then flick 30 times. Put it on the magnetic plate and shake for 1 min. Dump and add 1 ml 20% IFCS EC media (pre-warmed at 37 &#186;C).</li>
	<li>Shake vigorously 30 times then flick 30 times. Adjust pipet to 500 &#181;l and pipet up and down 10 times. Transfer 500 &#181;l to each chamber.</li>
	<li>Put at 37 &#186;C incubator and leave O/N. Next day, wash cells with 10% FBS EC media.</li>
</ol>
10. Preparing for Western Blot (WB) Samples
<ol>
	<li>Take out the tubes with beads/cells after 1 hr of rotation. Put them in the magnetic plate and shake for 2 min. Dump supernatant and add 1 ml cold HBSS. Shake vigorously 30 times then flick 30 times. Repeat twice for 3 washes, but with 1 min shaking on the plate.</li>
	<li>After last wash, put tubes on magnetic plate and shake for 1 min. Remove buffer using pipette and lyse the cells for WB using a method previously described11,12. Collected lysate usually contains around 30 &#181;g of protein.</li>
</ol>
11. Imaging
<ol>
	<li>Wash the cells 3 times with media, with final volume of 250 &#181;l for a chamber. Add 1.5 &#181;l of Dil-acLDL to the cells. From this point, keep the cells in the dark.</li>
	<li>Incubate for 3.5 hr at 37 &#730;C. Add 1.5 &#181;l of Hoechst, mix well and incubate for 30 min at 37 &#730;C.Wash cells with PBS and fix with 4% Paraformaldehyde (PFA) for 20 min at RT.</li>
	<li>Wash twice with PBS and block with 5% BSA-PBS for 30 min at RT. Wash twice with 1% BSA-PBS. Dilute BS-lectin-FITC using 1% BSA-PBS to a concentration of 2 &#181;g/ml and add 300 &#181;l to each chamber.</li>
	<li>Put the chamber on the orbital shaker at RT for 30 min. Wash with PBS 3 times.</li>
	<li>Visualize cells under the fluorescent microscope with exposure time of 200 msec for FITC (Lectin staining, an EC marker, excitation/emission 488 nm/519 nm), 30 msec for DAPI (Hoechst, a nuclear staining, excitation/emission at 358 nm/461 nm), and 300 msec for TRITC (acLDL staining, an EC marker, excitation/emission at 557 nm/576 nm). Acquire images using a 20X objective lens.</li>
</ol>

<ol>
	<li>Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+functions.">Endothelial cell functions.</a> J Cell Physiol. 196, 430-443 (2003).</li><li>Sumpio, B. E., Riley, J. T., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+in+focus:+endothelial+cell.">Cells in focus: endothelial cell.</a> Int J Biochem Cell Biol. 34, 1508-1512 (2002).</li><li>Cines, D. B., 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=Endothelial+cells+in+physiology+and+in+the+pathophysiology+of+vascular+disorders.">Endothelial cells in physiology and in the pathophysiology of vascular disorders.</a> Blood. 91, 3527-3561 (1998).</li><li>Deanfield, J. E., Halcox, J. P., Rabelink, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+function+and+dysfunction:+testing+and+clinical+relevance.">Endothelial function and dysfunction: testing and clinical relevance.</a> Circulation. 115, 1285-1295 (2007).</li><li>Jin, Y., Liu, Y., Antonyak, M., Peng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+vascular+endothelial+cells+from+murine+heart+and+lung.">Isolation and characterization of vascular endothelial cells from murine heart and lung.</a> Methods Mol Biol. 843, 147-154 (2012).</li><li>Makino, A., Platoshyn, O., Suarez, J., Yuan, J. X., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+connexin40+is+associated+with+coronary+endothelial+cell+dysfunction+in+streptozotocin-induced+diabetic+mice.">Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice.</a> Am J Physiol Cell Physiol. 295, C221-C230 (2008).</li><li>Marelli-Berg, F. M., Peek, E., Lidington, E. A., Stauss, H. J., Lechler, R. I. <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+murine+tissue.">Isolation of endothelial cells from murine tissue.</a> J Immunol Methods. 244, 205-215 (2000).</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 Protoc. 3, 1085-1091 (2008).</li><li>Nolan, D. J., 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=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev Cell. 26, 204-219 (2013).</li><li>Sobczak, M., Dargatz, J., Chrzanowska-Wodnicka, 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+and+culture+of+pulmonary+endothelial+cells+from+neonatal+mice.">Isolation and culture of pulmonary endothelial cells from neonatal mice.</a> J Vis Exp. , (2010).</li><li>Makino, A., Dai, A., Han, Y., Youssef, K. D., Wang, W., Donthamsetty, R., Scott, B. T., Wang, H., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=O-GlcNAcase+overexpression+reverses+coronary+endothelial+cell+dysfunction+in+type+1+diabetic+mice.">O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.</a> Am J Physiol Cell Physiol. 309, C593-C599 (2015).</li><li>Sasaki, K., Donthamsetty, R., Heldak, M., Cho, Y., Scott, B. T., Makino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VDAC:+old+protein+with+new+roles+in+diabetes.">VDAC: old protein with new roles in diabetes.</a> Am J Physiol Cell Physiol. 303, C1055-C1060 (2012).</li></ol>

The authors have nothing to disclose.

The most widely used endothelial cells in research are human umbilical vein endothelial cells (HUVECs) because of their convenience and ease of culturing. However, a lot of research requires the availability of a physiological model attained by the isolation of endothelial cells from other specific organs10. Endothelial cells make up a very minor population of cells in the heart tissue; their isolation and culturing can prove to be very difficult.
There are three critical steps with...

Mouse models of various cardiovascular diseases and metabolic disorders bear physiological and molecular changes which are similar to those found in patients. Furthermore, genetic alteration of mice is a powerful tool that allows us to investigate the pathogenic role of specific genes in the development and progression of diseases. Nowadays, cell-type-specific gene-knock-in or -out mice are easily generated in many laboratories and the measurement of mRNA and protein levels in specific cell types is the first step in determining whether the mice were truly genetically modified.
Endothelium is a thin single layer of endothelial cells (ECs) that lines the inner vascular wall. Endothelial cells play a critical role in regulating vascular tension, vascular permeability, and new vascular formation1,2. Endothelial dysfunction is a hallmark of many pathological conditions and changes in endothelial function can lead to various cardiovascular diseases3,4. It is thus significant to study the function of endothelial cells under physiological and pathophysiological conditions.
There are several ways to isolate ECs5-8 and the isolation method has to be optimized depending on which tissues and species will be used. This is because ECs from different tissues display a high level of heterogeneity with respect to their surface markers and protein expression9. Successful isolation of endothelial cells can often be challenging and requires some degree of training and practice. Once achieved, the method of isolation proposed in this protocol proves to be stable and efficient.
The overall goal of this method is to obtain mouse coronary ECs (MCECs) of high quality and quantity. Even though the quantity of cells collected from a heart is less than cells collected from other tissues using other techniques, this technique still provides better quality. The purity of endothelial cells in the resulting population is greater than 90%. Thus, this technique would be ideal for applications that rely on purely isolated endothelial cells such as cell imaging.

<table border="0" cellpadding="0" cellspacing="0" width="876">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:239px;">BSA</td>
			<td style="width:251px;">Sigma</td>
			<td style="width:108px;">A3311-10G</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Fisher</td>
			<td>BP120-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Fisher</td>
			<td>SH3026801</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H4034-500G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-(+)-Glucose&#160;</td>
			<td>Sigma</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Sheep anti-rat IgG beads&#160;</td>
			<td>Life Technologies</td>
			<td>F-410L</td>
			<td style="width:280px;">DynaBeads pan mouse IgG , Thermo Fisher Scientific, cat# 11035</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat anti-mouse PECAM Antibody&#160;</td>
			<td>BD Pharmigen</td>
			<td>550274</td>
			<td>BD Biosciences</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IFCS&#160;</td>
			<td>Fisher</td>
			<td>SH30072.04</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199</td>
			<td>Fisher</td>
			<td>MT10060-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dispase (Neutral Protease)</td>
			<td>Worthington Biochemical Corp./Fisher</td>
			<td>LS02109</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase Type II&#160;</td>
			<td>Worthington Biochemical Corp./Fisher</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol&#160;</td>
			<td>Fisher</td>
			<td>BP381-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Igepal</td>
			<td>Sigma</td>
			<td>13021-50ml</td>
			<td>I3021-50 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphatase inhibitor cocktail</td>
			<td>Sigma</td>
			<td>P0044-1ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protease inhibitor cocktail</td>
			<td>Sigma</td>
			<td>P8340-1 ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin&#160;</td>
			<td>Sigma</td>
			<td>H3149-100KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS&#160;</td>
			<td>Fisher/Mediatech</td>
			<td>MT35010CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Valine</td>
			<td>Sigma</td>
			<td>V1255-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGS</td>
			<td>BD</td>
			<td>356006</td>
			<td>Corning</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Fisher/Mediatech</td>
			<td>MT-30-002-Cl</td>
			<td>MT-30-002-CI</td>
		</tr>
	</tbody>
</table>

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.

The successful isolation of coronary endothelial cells from a mouse heart typically yields around 104 cells with over 90% purity with a cobblestone shape. For a pure population of endothelial cells, caution should be taken throughout the protocol to ensure a sterile environment free from any contamination. The appearance of elongated cells or enlarged cells within the population indicates contamination with other cell types, smooth muscle cells and fibroblasts. Endothelial cell...

Watch this Scientific Journal Video about Isolation of Mouse Coronary Endothelial Cells at JoVE.com

Isolation of Mouse Coronary Endothelial Cells

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-of-mouse-coronary-endothelial-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.1K Views.  University of Arizona, Tucson. 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.

Video: Isolation of Mouse Coronary 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 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 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 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.

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 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 ...

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 ...

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 Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...