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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+arterioles+in+type+2+diabetic+(db/db)+mice+undergo+a+distinct+pattern+of+remodeling+associated+with+decreased+vessel+stiffness.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+angiotensin+receptor+blocker+losartan+reduces+coronary+arteriole+remodeling+in+type+2+diabetic+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+coronary+endothelial+and+smooth+muscle+cells+from+A1+adenosine+receptor-knockout+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+attenuates+contraction+of+primary+cultured+rat+airway+smooth+muscle+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+two-phase+Ca2++release+and+apoptosis+controlled+by+TRP-ML1+channel+activity+in+coronary+arterial+myocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+heart+perfusion+according+to+Langendorff---still+viable+in+the+new+millennium.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+vascular+smooth+muscle+cell+differentiation+in+development+and+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SM22+alpha,+a+marker+of+adult+smooth+muscle,+is+expressed+in+multiple+myogenic+lineages+during+embryogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblast+to+myofibroblast+differentiation+in+vivo+and+in+vitro:+expression+of+focal+adhesion+components+in+neonatal+and+adult+rat+ventricular+myofibroblasts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+smooth+muscle+phenotypic+diversity+and+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+phenotype+and+function+of+vascular+smooth+muscle+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenteric+resistance+arteries+in+type+2+diabetic+db/db+mice+undergo+outward+remodeling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diameter+of+coronary+arteries+in+36+species+of+mammalian+from+mouse+to+giraffe.">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 bed specific factors. For example, compared to peripheral vessels, coronary arteries display different functional responses, exhibit distinct patterns of growth in responses to injury and display a unique array of channels, receptors and signaling molecules10,11. The coronary bed is also exposed to unique hemodynamic forces, since the vessels are compressed during systole by the contracting myocardium. Our recent work suggests that coronary microvascular remodeling in type 2 diabetic db/db mice differed from aortic and mesenteric resistance vessels1,12. To understand molecular mechanisms that account for these differences, in vitro studies using low passage VSMCs are necessary. Although there are a variety of different and well-established methods for isolating and culturing murine aortic or mesenteric artery VSMCs, currently, there is only one study that has been published on the isolation of VSMCs from the mouse coronary circulation3.
In this protocol, we demonstrate an improved method for isolation of primary murine VSMCs from the coronary circulation. Our protocol is loosely based on one published by Teng et al.3. The modifications we have applied to the original technique provide an enriched population and higher yield of VSMCs. The critical steps necessary to achieve these results include correct placement of the cannula, gentle flushing of the heart to remove excess blood, and cutting the apex of the heart at the end of digestion. Of the three, placement of the cannula is of the utmost importance. The coronary circulation is perfused from the coronary ostia just distal to the aortic valve. Therefore disruption of valve integrity via cannula insertion into the left ventricular chamber will lead to poor coronary perfusion and a dramatic reduction in the number of viable VSMCs. Gentle flushing of the coronary circulation will not only remove excess blood from the final pellet used for culture, but will also determine whether correct placement of the cannula is achieved. Finally, during digestion, some VSMCs may become trapped inside of the ventricles, thus cutting the apex at the conclusion of digestion will release the cells and yield a greater confluence at plating.
An important modification that can be utilized is the use of a 25 G gavage needle or a Harvard Apparatus mouse aorta cannula in place of the needle cannula. However, when using the gavage needle, one must take extra care not to block the coronary ostia with the ball at the end of the needle. The gentle flushing step will reveal if adjustment of the needle is necessary prior to perfusion. In addition, the type of collagenase used in the digestion solution is important for optimal VSMC isolation. Type II collagenase is not pure&#160;&#8211; it contains other enzymes such as trypsin, clostripain, and caseinase. Therefore, the amount of soybean trypsin inhibitor added to the digestion solution may need to be adjusted based on the specific lot of collagenase utilized in digestion.
One limitation of the technique is that VSMCs from the entire coronary circulation are isolated. Our previous studies focused on the coronary microvessels (80-120 &#956;m in diameter)1,2, however the main coronary arteries in mice are larger (&#62;160 &#956;m)13. In addition, this technique also isolates cells from the coronary venous circulation, not just the arterial circulation.
While the Langendorff technique has been used for over 100 years, the more recent advancements of mouse genome manipulation (e.g. knock-in, knock-out, mutations) provide a unique opportunity to utilize our updated technique for a wide variety of applications. After isolation and culture, the cells can be used for additional experiments including viral infection and treatment with pharmacological agents. The joint usage of genetically modified mice and standard cell culture will allow for deeper investigation of VSMCs from the murine coronary circulation.

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

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 possibility of contamination by adventitial and interstitial fibroblasts. To rule out this potential confound, we stained isolated cells for SM22&#945;, &#945;-smooth muscle actin (&#945;-SMA), both VSMC markers7,8, and vimentin, a fibroblast marker9, at passages 1-2 (Figure 2). We observed no change of these markers through passage 2. We also show that our cell isolation is devoid of CD31/PECAM staining (human coronary microvascular endothelial cells used as a positive control), indicating the lack of endothelial cell contamination using this isolation protocol. Collectively, our cell isolation procedure yields a nearly pure population of VSMCs from the coronary circulation and can now be used for further experimentation.
<img alt="Figure 1" src="/files/ftp_upload/53983/53983fig1.jpg" /> 
Figure 1: Representative cultured coronary VSMCs. Cultured coronary VSMCs (passage 2) isolated from heterozygous Db/db mice imaged at 10X magnification under bright field phase contrast conditions. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53983/53983fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53983/53983fig2.jpg" /> 
Figure 2: Immunofluorescence staining of cultured coronary VSMCs. Plated coronary VSMCs from heterozygous (Db/db) mice at passages 1-2 labeled with antibodies for SM22&#945;, &#945;-SMA, vimentin, and CD31/PECAM. Images taken at 10X magnification. No significant change was observed with passage. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53983/53983fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Murine Coronary Vascular Smooth Muscle Cells at JoVE.com

Isolation of Murine Coronary Vascular Smooth Muscle Cells

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

14.0K Views.  The Ohio State University College of Medicine.  Het doel van dit protocol is de isolatie en kweektechnieken van murine primaire vasculaire gladde spiercellen (VSMC's) van de coronaire circulatie demonstreren. Zodra VSMC's werden geïsoleerd, kunnen ze worden gebruikt voor vele standaard kweektechnieken. 

Video: Isolation of Murine Coronary Vascular Smooth Muscle Cells

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

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.

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

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

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

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

Neuroscience

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

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

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

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

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

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.

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation of Primary Mouse Lung Endothelial Cells

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

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

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

Immunology and Infection

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

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

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Medicine

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 of Mouse Coronary Endothelial Cells

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

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

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

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

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

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.

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of ...

Isolation and Magnetic Separation of Murine CD34+ Stem Cells from the Vascular Wall

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

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

Author Spotlight: Enhanced Isolation and Characterization of Vascular Wall Resident Murine CD34+ Stem Cells Using Magnetic Bead Screening-Coupled Flow Cytometry

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.

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

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