Name: Isolation of Murine Coronary Vascular Smooth Muscle Cells (Video) | JoVE Journal
Uploaded: 2016-05-30T13:00:00.000+00:00
Duration: 8 min 24 s
Description: Learn to isolate murine coronary vascular smooth muscle cells for cardiovascular research using perfusion-based techniques. This method yields a pure population suitable for standard cell culture assays.

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5 minutes

This work was supported by the National Institutes of Health (R01HL056046 to PAL and K99HL116769 to AJT), and The Research Institute at Nationwide Children&#39;s Hospital (to PAL and AJT).

Ethics Statement: This study was conducted in accordance with the National Institutes of Health Guidelines, and it was approved by the Institution Animal Care and Use Committee at Nationwide Children&#39;s Hospital.
1. Preparation/Set up
Note: This isolation technique requires two Langendorff heating coils positioned side-by-side on a ring stand, and connected in parallel to a circulating water bath.
<ol>
	<li>Turn on circulating water bath and adjust temperature to achieve a final temperature of 33-34 &#176;C at the tip of the Langendorff cannula (the water bath used in this set up is set to 40 &#176;C due to a 6-7 &#176;C loss in temperature across tubing). Turn on the perfusion pumps and set perfusion rate to 0.5 ml/min.
	<ol>
		<li>Clean both of the water-jacketed heating coils, attached tubing and 25 gauge cannula by flushing each with 50 ml of 70% ethanol, followed by 100 ml of autoclaved ultrapure water.</li>
		<li>Clear the heating coils by pushing air with a syringe and wash with 10 ml of Hank&#39;s Balanced Salt Solution (HBSS) without phenol red. Leave HBSS in the heating coils and tubing.</li>
		<li>Attach a sterile 10 ml luer-lock syringe filled with room temperature HBSS without phenol red to the tubing. Ensure that no air bubbles are trapped inside the tubing and heating coils when attaching the HBSS-filled syringe as this could cause air emboli during the digestion to negatively impact the VSMC isolation.</li>
	</ol>
	</li>
	<li>Remove the 25 gauge cannula from the heating coils and place on another sterile 10 ml syringe filled with ice-cold HBSS without phenol red. Keep this on ice until ready to use. Repeat for the other cannula.</li>
	<li>Prepare Enzyme Digestion Solution.
	<ol>
		<li>Weigh collagenase type 2 according to specific lot number for 75 ml of digestion solution to achieve a final concentration of 300 Units/ml.</li>
		<li>Dissolve collagenase in 75 ml of HBSS with phenol red. Next, add 1.5 ml of 0.1 mg/ml soybean trypsin inhibitor and 75 &#181;l of 1 M CaCl2 to the mixture.</li>
	</ol>
	</li>
	<li>If using the digestion solution immediately, warm it to 37 &#176;C. If not, place it at 4 &#176;C for up to 6 hr. This solution must be made fresh daily.</li>
	<li>Prepare the stop solution/plating media.
	<ol>
		<li>Add 100 ml heat-inactivated FBS, 5 ml of L-glutamine, 5 ml of non-essential amino acids, 5 ml of HEPES, and 1 ml of primocin to 500 ml of high glucose (4.5 g/L) DMEM. Filter sterilization is recommended.</li>
		<li>Aliquot 6 ml of the stop solution into each of twelve 15 ml conical tubes, numbered A1-A6 and B1-B6, and place at 4 &#176;C. Aliquot 50 ml of stop solution into a 50 ml conical and place in 37 &#176;C incubator. 
		Note: This step can also be prepared the day prior to isolation if necessary. This stop solution will be used as the plating media following cell isolation.</li>
	</ol>
	</li>
	<li>Prepare two 1 ml syringes equipped with a 30 G needle with 100 &#181;l of heparin (1,000 units/ml).</li>
	<li>Cut a 5 cm piece of 5-0 surgical silk. Tie a loose overhand knot in the surgical silk with one end twice as long as the other. Place onto the 25-gauge cannula, but do not tighten. Repeat for the other 25 G cannula.</li>
	<li>Fill four 35 mm sterile Petri dishes (#1-4) with 2 ml of sterile, ice-cold, HBSS without phenol red. Keep on ice.</li>
</ol>
2. Euthanasia, Heart Isolation and Cannulation
<ol>
	<li>Before heart isolation, ensure that the digestion solution and 50 ml conical of stop solution are warm. Remove the HBSS-filled syringe with attached 25 G cannula from the ice and place in a burette clamp (or ring stand clamp) on a ring stand.</li>
	<li>Anesthetize mice using 3% isofluorane. Confirm the mouse is anesthetized by a toe touch on each hind limb. If the leg twitches, the mouse is not yet anesthetized so wait 1 min and repeat the toe touch.</li>
	<li>Expose the jugular vein by removing the fur and skin on the anterior of the neck with scissors. Next, use forceps to carefully remove any fat surrounding the jugular vein. After removing fat, slowly inject 100 &#181;l of heparin (1,000 units/ml). Place a surgical sponge over the vein to avoid excessive bleeding.</li>
	<li>After 1 min, open chest to expose heart, lift heart with curved forceps and excise it using scissors, making sure to keep the ascending aorta intact through the first brachiocephalic branch. Immediately place heart into a 35 mm dish (#1) with ice-cold HBSS without phenol red and rinse. 
	Note: Perform the following 4 steps using a dissecting scope.</li>
	<li>Position the 25 G cannula that is mounted on the ring stand at a 45&#176; angle so that the tip is located just under the surface of ice-cold HBSS in dish #2 and is clearly visible through the dissecting microscope. Transfer the heart to dish #2.</li>
	<li>Using Dumont forceps, expose the aorta by gently removing excess adipose via blunt dissection to prevent puncturing or tearing of the aorta. Next, use forceps to slide the aorta over the 25 G cannula.</li>
	<li>Guide the cannula downward through the aorta, close to, but not beyond the aortic valve, which will compromise the integrity of the valve and therefore, the retrograde perfusion. Once the cannula is in place, slide the pre-tied silk over the aorta, and tighten. Tie a second knot to form a square knot around the aorta to secure the heart.</li>
	<li>Once the aorta is securely tied, gently flush the remaining blood from the coronary circulation with the ice-cold HBSS in the syringe attached to the 25 G cannula. Take care to not push too much to avoid excessive coronary pressure. Only a very slow and gentle flush is required to complete the flush. Leak-free aortic cannulation can also be observed during this gentle flush.</li>
	<li>Turn on the perfusion pump to begin the flow of HBSS. Remove the 25 G cannula (with the heart attached) from the HBSS-filled syringe, taking care to keep the cannula hub filled with HBSS, and connect it to the warm HBSS-filled heating coils. Begin perfusion of the heart with the perfusion pump at a rate of 0.5 ml/min for 8 min.</li>
	<li>After the first heart is cannulated and is being perfused on the system, repeat steps 2.1-2.8 for the second heart. These isolations will be staggered for each heart therefore careful planning and timing are required.</li>
</ol>
3. Digestion
<ol>
	<li>After flushing with HBSS, change the 10 ml HBSS syringe currently on the pump to one filled with 10 ml of pre-warmed digestion solution. Perfuse each heart with digestion solution for 12 min at a rate of 0.5 ml/min. Do not collect fractions stemming from this first digestion as this will contain any remnant blood and endothelial cells.</li>
	<li>After 12 min, change the perfusion rate to 0.4 ml/min, place a 15 ml conical (tube A1 or B1, corresponding to the first or second heart for each digestion) filled with cold stop solution into an ice bucket beneath the heart in order to begin VSMC-enriched perfusate collection.</li>
	<li>After 15 min, change the collection conical to tube A2 or B2 and spin conical tube A1 and B1 at 89 x g for 10 min immediately after collection of both fractions. When the syringe filled with the digestion solution on the pump has less than 1 ml of solution, replace with a freshly-filled 10 ml syringe of digestion solution.</li>
	<li>After centrifugation, aspirate supernatant from each collection tube, leaving approximately 0.5 ml. Resuspend pellet from A1 with 2 mL of warm stop solution (or plating media). Add resuspension from A1 tube to B1 tube and place tube in 37 &#176;C incubator with the cap loose.</li>
	<li>Repeat steps 3.3 and 3.4 until all the tubes (through A6 and B6) are used for a total collection time of 90 min. At 80 min of digestion, cut open the heart apex to flush out any cells that may be trapped inside the ventricle and collect these cells in tube A6 and B6, respectively. 
	Note: Occasionally the heart will fall off the cannula prior to the 90 min time point. If this occurs, retrieve the heart and place into a sterile 35 mm Petri dish filled with 2 ml of digestion solution. Cut the apex of the heart and gently rinse the heart with digestion solution. Next, filter the 2 ml of digestion solution through a sterile 100 &#181;m cell strainer into a 50 ml conical and add to tube A6 or B6.</li>
	<li>After each spin of subsequent tubes (A2 and B2), add the resuspended VSMCs to the previous collection tube (combined A1 and B1). Also, take care not to let the digestion solution in the pump run out and switch the 10 ml syringes as necessary throughout the digestion.</li>
	<li>After all the tubes are combined, centrifuge the VSMC-rich suspension at 89 x g for 10 min.</li>
	<li>Aspirate as much supernatant from collection tubes as possible. Resuspend the remaining pellet in 2 ml of plating media and plate on one 35 mm culture dish. Place culture dish in 37 &#176;C incubator for 24 hr.</li>
</ol>
4. Cell Culture
Note: Complete the remaining steps in the biosafety cabinet.
<ol>
	<li>After 24 hr, gently aspirate the plating media. The culture will normally have a large amount of debris. To wash the cells, add 2 ml of warm, sterile PBS and gently tap the dish against two fingers while rotating in a clockwise motion.</li>
	<li>After rotating 360&#176;, aspirate the PBS and repeat a second time. Aspirate the PBS and add another 2 ml of sterile, warm PBS. Check the culture dish under the microscope to make sure there is little to no debris on the plate. Cell confluence can also be determined at this time.</li>
	<li>If debris remains in the culture dish, repeat step 4.2. If the culture dish is clear of debris, aspirate the PBS and add 2 ml of warm plating media.</li>
	<li>After 24 hr, wash the cells again with warm, sterile PBS to remove any final debris or dead cells from the culture. Aspirate the PBS and place another 2 ml of warm plating media on the cells.</li>
	<li>Check on the cells every day and feed every 48 hr with fresh warmed plating media. At this point, cells can be used for standard cell culture assays. VSMCs should be approximately 80% confluent 4-5 days after plating, although may take up to 7 days until the procedure is perfected. If splitting to subsequent passages, use a maximum of 1:4 35 mm dishes (or to a comparable area of culture dishes).</li>
</ol>

<ol><li>Katz, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coronary+arterioles+in+type+2+diabetic+%28db%2Fdb%29+mice+undergo+a+distinct+pattern+of+remodeling+associated+with+decreased+vessel+stiffness%5BTitle%5D)+AND+%22Basic+Res+Cardiol%22%5BJournal%5D)">Coronary arterioles in type 2 diabetic (db/db) mice undergo a distinct pattern of remodeling associated with decreased vessel stiffness.</a> Basic Res Cardiol. 106 (6), 1123-1134 (2011).</li>
<li>Husarek, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+angiotensin+receptor+blocker+losartan+reduces+coronary+arteriole+remodeling+in+type+2+diabetic+mice%5BTitle%5D)+AND+%22Vascul+Pharmacol%22%5BJournal%5D)">The angiotensin receptor blocker losartan reduces coronary arteriole remodeling in type 2 diabetic mice.</a> Vascul Pharmacol. , (2015).</li>
<li>Teng, B., Ansari, H. R., Oldenburg, P. J., Schnermann, J., Mustafa, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+coronary+endothelial+and+smooth+muscle+cells+from+A1+adenosine+receptor-knockout+mice%5BTitle%5D)+AND+%22Am+J+Physiol+Heart+Circ+Physiol%22%5BJournal%5D)">Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice.</a> Am J Physiol Heart Circ Physiol. 290 (4), H1713-H1720 (2006).</li>
<li>Oldenburg, P. J., Wyatt, T. A., Sisson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ethanol+attenuates+contraction+of+primary+cultured+rat+airway+smooth+muscle+cells%5BTitle%5D)+AND+%22Am+J+Respir+Cell+Mol+Biol%22%5BJournal%5D)">Ethanol attenuates contraction of primary cultured rat airway smooth muscle cells.</a> Am J Respir Cell Mol Biol. 43 (5), 539-545 (2010).</li>
<li>Xu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+two-phase+Ca2%2B+release+and+apoptosis+controlled+by+TRP-ML1+channel+activity+in+coronary+arterial+myocytes%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">Intracellular two-phase Ca2+ release and apoptosis controlled by TRP-ML1 channel activity in coronary arterial myocytes.</a> Am J Physiol Cell Physiol. 304 (5), C458-C466 (2013).</li>
<li>Skrzypiec-Spring, M., Grotthus, B., Szelag, A., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolated+heart+perfusion+according+to+Langendorff---still+viable+in+the+new+millennium%5BTitle%5D)+AND+%22J+Pharmacol+Toxicol+Methods%22%5BJournal%5D)">Isolated heart perfusion according to Langendorff---still viable in the new millennium.</a> J Pharmacol Toxicol Methods. 55 (2), 113-126 (2007).</li>
<li>Owens, G. K., Kumar, M. S., Wamhoff, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+regulation+of+vascular+smooth+muscle+cell+differentiation+in+development+and+disease%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Molecular regulation of vascular smooth muscle cell differentiation in development and disease.</a> Physiol Rev. 84 (3), 767-801 (2004).</li>
<li>Li, L., Miano, J. M., Cserjesi, P., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SM22+alpha%2C+a+marker+of+adult+smooth+muscle%2C+is+expressed+in+multiple+myogenic+lineages+during+embryogenesis%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">SM22 alpha, a marker of adult smooth muscle, is expressed in multiple myogenic lineages during embryogenesis.</a> Circ Res. 78 (2), 188-195 (1996).</li>
<li>Santiago, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+fibroblast+to+myofibroblast+differentiation+in+vivo+and+in+vitro%3A+expression+of+focal+adhesion+components+in+neonatal+and+adult+rat+ventricular+myofibroblasts%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts.</a> Dev Dyn. 239 (6), 1573-1584 (2010).</li>
<li>Fisher, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascular+smooth+muscle+phenotypic+diversity+and+function%5BTitle%5D)+AND+%22Physiol+Genomics%22%5BJournal%5D)">Vascular smooth muscle phenotypic diversity and function.</a> Physiol Genomics. 42A (3), 169-187 (2010).</li>
<li>Archer, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diversity+of+phenotype+and+function+of+vascular+smooth+muscle+cells%5BTitle%5D)+AND+%22J+Lab+Clin+Med%22%5BJournal%5D)">Diversity of phenotype and function of vascular smooth muscle cells.</a> J Lab Clin Med. 127 (6), 524-529 (1996).</li>
<li>Souza-Smith, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenteric+resistance+arteries+in+type+2+diabetic+db%2Fdb+mice+undergo+outward+remodeling%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Mesenteric resistance arteries in type 2 diabetic db/db mice undergo outward remodeling.</a> PLoS One. 6 (8), e23337(2011).</li>
<li>Thuroff, J. W., Hort, W., Lichti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diameter+of+coronary+arteries+in+36+species+of+mammalian+from+mouse+to+giraffe%5BTitle%5D)+AND+%22Basic+Res+Cardiol%22%5BJournal%5D)">Diameter of coronary arteries in 36 species of mammalian from mouse to giraffe.</a> Basic Res Cardiol. 79 (2), 199-206 (1984).</li>
</ol>

The authors declare that they have no competing financial interests.

The purpose of this study was to adapt existing cell isolation protocols to increase the yield of coronary vascular smooth from murine hearts. Most of the pioneering work in vascular smooth muscle biology was performed with cultured rat aortic smooth muscle cells. These studies provided fundamental knowledge of molecular mechanisms that control VSMC growth, migration and hypertrophy7. However, as the field progressed, it became apparent that VSMC phenotype and function was controlled by a number of vascular be...

The goal of this method is to isolate vascular smooth muscle cells (VSMCs) from the murine coronary circulation for use in cell culture and standard cell culture assays. We developed this technique to assess the molecular mechanisms of vascular remodeling in diabetes. We have previously reported inward hypertrophic remodeling in the septal coronary arterioles in the db/db mouse model of diabetes1. Due to the limited amount of tissue found in the murine septal coronaries, standard experimental techniques investigating protein changes (e.g. western blot) in db/db and control mice are difficult at best. In addition, we have previously shown that the angiotensin receptor blocker (ARB) losartan reduces the remodeling observed in db/db mice2. Therefore, isolation of primary VSMCs from the coronary circulation allows us to further investigate changes in VSMC phenotype or activated signaling pathways in diabetic mice, which may be contributing to adverse coronary arteriole remodeling.
Numerous studies have elucidated canonical signaling pathways using VSMCs isolated from rodent aorta, rather than in each specific vascular bed. However, we have demonstrated vascular-bed specific remodeling in coronary, aortic, and mesenteric circulations of db/db mice1, suggesting the VSMCs in each vascular bed may be different. Therefore, it is necessary to isolate VSMCs from each vascular bed in order to better understand the pathological changes occurring in each set of VSMCs. There are a plethora of different methods for isolating and culturing aortic VSMCs. However, currently, there is only one study that has been published on the isolation of VSMCs from the mouse coronary circulation3. Teng et al. was the first to report a method for isolating VSMCs from the mouse coronary circulation; however, we have amended the protocol significantly as they also isolated endothelial cells. Other labs have also used the protocol from Teng et al. to isolate coronary arterial myocytes and airway smooth muscle cells4,5. The alterations we have incorporated will yield a population of cells highly enriched for VSMCs from the coronary circulation.
The retrograde perfusion of the isolated mammalian heart, or Langendorff technique, was established in 18976 by Oscar Langendorff and is still widely used today for the isolation of cardiovascular cells. The technique presented here, coupled with the advancement of modern murine genetic modifications, provides a valuable tool for closer investigation of the molecular behavior of VSMCs from the coronary circulation.

<table><tbody><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>16140-071</td><td></td></tr><tr><td>HEPES 1 M solution</td><td>Fisher</td><td>MT-25-060</td><td></td></tr><tr><td>Primocin - 20 ml</td><td>Invivogen</td><td>ant-pm-2</td><td></td></tr><tr><td>DMEM (High Glucose, Sodium Pyruvate, L-Glutamine)&amp;nbsp;</td><td>Life Technologies</td><td>11995-065</td><td></td></tr><tr><td>MEM NEAA 10 mM 100x</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>L-Glut 200 mM - Gibco</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>Sterile Cell Strainer 100 &amp;micro;m nylon mesh</td><td>Fisher</td><td>22363549</td><td></td></tr><tr><td>Nunclon Polystrene dish with lid, sterile, 35 mm</td><td>Fisher</td><td>12-565-91</td><td></td></tr><tr><td>Harvard Apparatus black silk suture 5-0</td><td>Fisher</td><td>14-516-124</td><td></td></tr><tr><td>Collagenase Type-2&amp;nbsp;</td><td>Worthington Biochemical</td><td>LS004176</td><td></td></tr><tr><td>Soybean Trypsin Inhibitor 25 mg</td><td>Sigma</td><td>T6522</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS) (1x), liquid (clear)</td><td>Life Technologies</td><td>14175-103</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS) (1x), liquid (phenol red)</td><td>Life Technologies</td><td>14170-161</td><td></td></tr><tr><td>5.0 ml heating coil with degassing bubble trap</td><td>Radnoti</td><td>158830</td><td></td></tr><tr><td>11 plus pump</td><td>Harvard Apparatus</td><td>70-2208</td><td></td></tr><tr><td>Circulating heated water pump</td><td></td><td></td><td>any brand will work</td></tr></tbody></table>

isolation of murine coronary vascular smooth muscle cells

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.

Due to the novel aspect of our coronary VSMC isolation technique, we sought to determine the purity of the cell isolation. Mouse coronary VSMCs were identified based on their morphology and immunofluorescence staining up to passage 2. Based on morphology of the cells in culture after the first wash, the isolation procedure effectively removes cardiac myocytes and endothelial cells. The VSMCs retain their morphology up to passage 2 (Figure 1). However, there is a possibili...

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 ...

The overall goal of this procedure is to isolate vascular smooth muscle cells from the murine coronary circulation for their culture and use in standard cell culture assays. This method can help answer key questions in the cardiovascular field such as what key disease mechanisms are involved in murine primary coronary vascular smooth muscle cell behavior. The main advantage of this technique is that it yields a mostly pure population of mouse primary coronary vascular smooth muscle cells.Before beginning the procedure attach a sterile 10-milliliter Leur Lock syringe filled with room temperature HBSS without phenol red to the perfusion pump tubing. Next, transfer each of the 25-gauge cannulas from the heating coils onto individual 10-milliliter syringes filled with ice-cold HBSS without phenol red. Place the syringes on ice.Then, tie loose, overhand knots in each of two five-centimeter pieces of silk suture with one end twice as long as the other. Place one knot onto each of the 25-gauge cannulas without tightening and place one of the HBSS-filed syringes into a burette clamp on a ring stand. When all of the materials are ready, confirm a lack of response to toe touch and use scissors to remove the fur and skin on the anterior neck of the anesthetized experimental mouse.When the jugular vein is visible use forceps to carefully remove any fat surrounding the vein and slowly inject 100 microliters of heparin, placing a surgical sponge over the vein to avoid excessive bleeding. After one minute, open the chest to expose the heart. Using curved forceps to life the heart, excise the organ with scissors, taking care to keep the ascending aorta intact through the first brachiocephalic branch.Then, immediately rinse the heart in a 35-millimeter dish containing ice-cold HBSS without phenol red. Next, position the ring stand-mounted cannula at a 45-degree angle next to a dissecting microscope so that the tip is just under the surface of a second petri dish of ice-old HBSS and clearly visible through the objective. Transfer the heart under the microscope and use Dumont forceps to gently blunt dissect the excess adipose tissue to prevent the puncturing or tearing of the aorta.When all of the tissue has been removed, use forceps to slide the vessel over the cannula and guide the needle downward through the aortic lumen close to but not beyond the aortic valve. Once the cannula is in place, slide the pre-tied silk over the aorta and tighten the knot. Then, tie a second knot to form a square knot around the aorta, securing the heart.Now, slowly and gently flush the remaining blood from the coronary circulation with the ice-cold HBSS and turn on the perfusion pump to begin the flow of HBSS. Remove the cannula and heart from the syringe, taking care to keep the cannula hub filled with HBSS and connect the cannula to the warm HBSS-filled heating coils. Then, begin an eight-minute HBSS perfusion of the heart at a rate of 0.5 milliliters per minute.Immediately cannulating and perfusing the next heart as soon as the perfusion of the first heart has begun. After all of the HBSS has been perfused, replace the HBSS syringe with 10 milliliters of pre-warmed digestion solution and perfuse the hearts with the digestion solution at 0.5 milliliters per minute. After 12 minutes change the perfusion rate to 0.4 milliliters per minute and place 15-milliliter conical tubes filled with cold stop solution into an ice bucket beneath the hearts to begin the vascular smooth muscle cell-enriched perfusate collection.After 15 minutes replace the first set of collection tubes with a second set of tubes and immediately spin down both tubes containing the first vascular smooth muscle cell fraction. When less than one milliliter of digestion solution is left in the syringe, attach a new syringe containing 10 milliliters of fresh digestion solution. At the end of the centrifugation aspirate all but the last 0.5 milliliters of supernatant from each collection tube and resuspend the pellet from the first heart in two milliliters of warm stop solution.Next, mix the cell suspension from the first heart with the pellet from the second heart and store the pooled cells in a 37 degrees Celsius incubator with a loose cap. After 80 minutes of digestion, cut open the heart apex to flush out any of the remaining cells trapped inside the ventricle and collect these cells in the sixth set of collection tubes. When all of the cell fractions have been combined, spin down the pooled vascular smooth muscle cell rich suspensions and aspirate as much of the supernatant from the collection tubes from the two hearts as possible.Then, resuspend the pellets in two milliliters of plating medium and pool them in a 35-millimeter culture dish at 37 degrees Celsius for 24 hours of culture. The next day, gently aspirate the medium from the vascular smooth muscle cell culture in a biosafety cabinet. Then, wash the cells two times with two milliliters of warm sterile PBS gently tapping the dish against two fingers while rotating the plate in a clockwise motion for 360 degrees.After the second rinse, replace the wash with two milliliters of fresh warm sterile PBS and check the culture under the microscope to confirm confluency and the absence of debris. Replace the PBS with two milliliters of warm plating medium and return the cells to the incubator for 24 hours. The next day, wash the cells again to remove any final debris and dead cells from the culture.Then, replace the PBS with two milliliters of warm plating medium and return the cells to the incubator. As assessed by the morphology of the cells in the culture after the first wash the isolation procedure effectively removes cardiac myocytes and endothelial cells. To rule out the possibility of contamination by adventitial and interstitial fibroblasts, the isolated cells can be stained for SM22 alpha and alpha smooth muscle actin, both vascular smooth muscle cell markers, and vimentin, a fibroblast marker up through passage two.Staining for PCAM also confirms the lack of endothelial cell contamination, demonstrating that a nearly pure population of vascular smooth muscle cells can be isolated from the coronary circulation by this protocol. Once mastered, this entire technique can be completed in less than two hours if it is performed properly. After watching this video you should have a good understanding of how to isolate primary murine vascular smooth muscle cells.

isolation-of-murine-coronary-vascular-smooth-muscle-cells

Research

JoVE Journal

Biology

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

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.

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

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.

Isolation of Murine Valve 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.

Isolation of Mouse Coronary Endothelial Cells

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 "myovascular unit".

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Isolation and Purification of Murine Cardiac Pericytes

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

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.

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

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Learn to isolate murine coronary vascular smooth muscle cells for cardiovascular research using perfusion-based techniques. This method yields a pure population suitable for standard cell culture assays.

Isolation of Murine Coronary Vascular Smooth Muscle Cells

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