Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

Summary

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

Abstract

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Introduction

Calcified aortic valve disease (CAVD) is the most prevalent valvular heart disease in western populations, affecting nearly 2.5% of elderly individuals over 65 years of age¹. CAVD affects over six million Americans and is associated with changes in the mechanical properties of the leaflets that impair normal blood flow-through^1,2. Currently, there is no pharmacological treatment to stop the progression of the disease or to activate mineral regression. The only effective therapy to treat CAVD is aortic valve replacement by surgery or transcatheter aortic valve replacement

Protocol

NOTE: All animal procedures described here have been approved by Icahn School of Medicine at Mount Sinai institutional core and use committee.

1. Preparation before valve cell isolation from adult mice

1. Clean and sterilize all the surgical instruments shown in Figure 1A by using 70% v/v ethanol and subsequently autoclaving them for 30 min. clean the surgical workspace with 70% ethanol.
2. Add 500 µL of penicillin-streptomycin to 50 mL of 10 mm HEPES. Prepare an aliquot of 50 mL of 1x phosphate-buffered saline (PBS). Keep the solutions on ice.
3. Prepare 1 mg/mL and 4.5 mg/mL collag....

Discussion

This article presents a detailed protocol of mouse valve cell isolation for primary culture. Three aortic valves from 8-week-old mice were pooled to obtain an adequate number of cells. In addition, this protocol describes the characterization of VIC phenotype and the in vitro mineralization assay. The method was adapted from the previously described protocol from Mathieu et al.⁷.

During the isolation of aortic valves, care must be taken to avoid all so.......

Materials

Name	Company	Catalog Number	Comments
3 mm cutting edge scissors	F.S.T	15000-00
Anti-alpha smooth muscle Actin antibody	abcam
Anti-mouse, Alexa Fluor 488 conjugate	Cell Signaling	4412
Arsenazo-III reagent set	POINT SCIENTIFIC	C7529-500	a Kit to measure the concentration of calcium
Bonn Scissors	F.S.T	14184-09
Calcium hydroxide	SIGMA -Aldrich 31219	31219
CD31	Novusbio
Collagenase type I  (125 units/mg)	Thermofisher Scientific	17018029
DMEM	Tthermofisher	11965092
Extra fine graefe forceps	F.S.T	11150-10
FBS	Gibco 16000044
Fine forceps	F.S.T Dumont
HCl	SIGMA-ALDRICH	H1758
HEPES 1 M solution	STEMCELLS TECHNOLOGIES
L-Glutamine 100x	Thermofisher Scientific	25030081
Mycozap	Lanza	VZA-2011	Mycoplasma elimination reagent
PBS 10x	SIGMA-ALDRICH
penecillin streptomycin 100x	Thermofisher Scientific	10378016
Sodium Pyruvate 100 mM	Thermofisher Scientific	11360070
Standard pattern forceps	F.S.T	11000-12
Surgical Scissors - Sharp-Blunt	F.S.T	14008-14
Trypsin 0.05%	Thermofisher Scientific	25300054
Vimentin	abcam