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 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 SM22α, α-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 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.
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'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.
2. Euthanasia, Heart Isolation and Cannulation
3. Digestion
4. Cell Culture
Note: Complete the remaining steps in the biosafety cabinet.
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 SM22α, α-smooth muscle actin (α-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.
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 µm. Please click here to view a larger version of this figure.
Figure 2: Immunofluorescence staining of cultured coronary VSMCs. Plated coronary VSMCs from heterozygous (Db/db) mice at passages 1-2 labeled with antibodies for SM22α, α-SMA, vimentin, and CD31/PECAM. Images taken at 10X magnification. No significant change was observed with passage. Scale bars = 100 µm. Please click here to view a larger version of this figure.
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 – 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 μm in diameter)1,2, however the main coronary arteries in mice are larger (>160 μm)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 authors have nothing to disclose.
This work was supported by the National Institutes of Health (R01HL056046 to PAL and K99HL116769 to AJT), and The Research Institute at Nationwide Children's Hospital (to PAL and AJT).
Fetal bovine serum | Life Technologies | 16140-071 | |
HEPES 1M solution | Fisher | MT-25-060 | |
Primocin – 20mL | Invivogen | ant-pm-2 | |
DMEM (High Glucose, Sodium Pyruvate, L-Glutamine) | Life Technologies | 11995-065 | |
MEM NEAA 10 mM 100X | Life Technologies | 11140-050 | |
L-Glut 200 mM – Gibco | Life Technologies | 25030-081 | |
Sterile Cell Strainer 100um nylon mesh | Fisher | 22363549 | |
Nunclon Polystrene dish with lid, sterile, 35 mm | Fisher | 12-565-91 | |
Harvard Apparatus black silk suture 5-0 | Fisher | 14-516-124 | |
Collagenase Type-2 | Worthington Biochemical | LS004176 | |
Soybean Trypsin Inhibitor 25mg | Sigma | T6522 | |
Hanks' Balanced Salt Solution (HBSS) (1X), liquid (clear) | Life Technologies | 14175-103 | |
Hanks' Balanced Salt Solution (HBSS) (1X), liquid (phenol red) | Life Technologies | 14170-161 | |
5.0 ml heating coil with degassing bubble trap | Radnoti | 158830 | |
11 plus pump | Harvard Apparatus | 70-2208 | |
Circulating heated water pump | any brand will work |