Isolation of Murine Coronary Vascular Smooth Muscle Cells

Summary

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.

Abstract

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.

Introduction

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 a....

Protocol

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.

1. Turn on circulating water bath and adjust temperature to achieve a final temperature of 33-34 °C at the tip of the Langendorff cannula (the water bath used in this set up is set to 40 °C due to a 6-7 °C loss in temperature across t....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fetal bovine serum	Life Technologies	16140-071
HEPES 1 M solution	Fisher	MT-25-060
Primocin - 20 ml	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 100 µm 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 25 mg	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