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.
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.
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-through1,2. 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 replacement3. It is therefore imperative to investigate the molecular mechanisms leading to valve mineralization to identify new pharmacological targets. Indeed, non-treated aortic stenosis has several adverse consequences such as left ventricle dysfunction and heart failure4.
The aortic valve consists of three layers known as fibrosa, spongiosa, and ventricularis, which contain VICs as the predominant cell type5. The fibrosa and the ventricularis are covered by a layer of vascular endothelial cells (VECs)5. The VECs regulate the permeability of inflammatory cells as well as paracrine signals. Increased mechanical stress may affect the integrity of the VECs and disturb the homeostasis of the aortic valve, leading to inflammatory cell invasion6. Scanning electron microscopy analyses showed disrupted endothelium in a human calcified aortic valve7.
Histological analyses of calcified tissue reveal the presence of osteoblasts and osteoclasts. Furthermore, osteogenic differentiation of VICs was observed both in vitro and in human valve tissue8. This process is mainly orchestrated by the Runt-related transcription factor 2 (Runx2) and the bone morphogenetic proteins (BMPs)8,9.
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
2. Isolation of valve cells
3. Analysis of cell identity and morphology
NOTE: Immunofluorescence staining was used to study cell morphology and endothelial cell contamination.
4. In vitro calcification assay
As murine aortic valves are typically 1 mm in diameter, at least three valves must be pooled to collect a million viable cells for different experimental procedures. The different steps of the VIC isolation process are shown in Figure 1 and Figure 2. As it is difficult to manually scrape the valve tissue, it is preferable to use shear stress created by vortexing to remove the VECs. Indeed, the CD31 immunofluorescence staining results showed the absence of endothelial cells contamination (Figure 3D). In addition, mouse VICs express vimentin and α-SMA, which are the major markers of valve cells (Figure 3B,C).
Cell mineralization in vitro
A calcium reagent kit was used to measure the calcium concentration; cells treated with calcifying medium have higher calcium concentration compared to non-treated cells (Figure 4A). The concentration of calcium was normalized with the total protein concentration. Alizarin red staining confirmed the calcium-reagent kit measurements by showing red positive calcium nodes (Figure 4B).
Figure 1: Description of valve dissection. (A) Representative image of all the surgical instruments needed for the dissection, scissors 2 is needed to open the skin of the mouse and scissors 3 to open the chest. Tweezers 5 and 6 are needed to hold the skin and open the chest. (B) Leave 3 mm of tissue from the aorta (black arrow). (C) Cut the heart in the middle of the ventricles with scissors number 4. (D) Open the heart toward the aortic valve with scissors 3. Use the thin tweezers 7 and 8 to carefully dissect the aortic valve. The valve is visible and has some black dots that are characteristic of mice valve tissue (blue arrow). (E) Increase the magnification to better visualize the aortic valve. Isolate the valve with the small scissors 4; (F) maintain the tissue with tweezers 7. Please click here to view a larger version of this figure.
Figure 2: Representative description of mouse valve cell isolation. Abbreviations: HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; RT = room temperature; DMEM = Dulbecco's modified Eagle medium; FBS = fetal bovine serum. Please click here to view a larger version of this figure.
Figure 3: Mouse valve cell phenotype. Microscopic view of (A) freshly isolated valve cells. Immunofluorescence staining showing (B) vimentin-positive cells and (C) α-SMA. Cells are negative for (D) CD31 staining. Scale bars = 200 µm. Abbreviations: DAPI = 4′,6-diamidino-2-phenylindole; CD31 = cluster of differentiation 31; α-SMA = alpha-smooth muscle actin. Please click here to view a larger version of this figure.
Figure 4: In vitro calcification assay. (A) Phosphate-rich calcifying medium induced VIC calcification in vitro, which was measured with a reagent kit. (B) Microscopic image showing red positive staining (right) for calcium nodes. (C) Alizarin red staining showed positive calcium nodes (black arrow) of VICs in response to calcifying medium. Scale bars = 100 µm. Abbreviations: CTL- = Control; mVICs = mouse valvular interstitial cells. Please click here to view a larger version of this figure.
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 sources of possible contagion to protect the cells from bacterial or mycoplasma contamination. Indeed, it is crucial to autoclave all the surgical tools prior to starting the experiments. The HEPES solution should be supplemented with 1% antibiotics to minimize bacterial infection. Furthermore, mycoplasma may cause cytopathology and consequently interfere with every parameter measured in cell culture10.
Plating cells in small culture dishes with lower volume of culture medium is critical for VIC growth and proliferation. Letting the tissue settle and adhere to the cell culture dish permits cell migration from the tissue to the dish wall. Given that isolated cells from young mice proliferate faster, it is recommended to transfer cells to a larger culture dish of 75 cm2 after 5 days of culture. Maintaining cells to 80% confluence is crucial to minimize the differentiation of VICs to a myofibroblast phenotype8.
As shown by immunofluorescence imaging, the isolated valve cells show a fibroblast-like phenotype. VICs have an elongated cytoplasm and express both vimentin and αSMA as described by previous studies. The present work confirmed that the mouse VIC phenotype is similar to that previously described for porcine VICs11 and human VICs12. Most in vitro studies on aortic stenosis are performed on cells from large animals8,11. The key disadvantage of porcine VICs is their spontaneous differentiation to an osteoblast phenotype in vitro even in normal media13. However, mouse VICs do not calcify spontaneously even at higher passages.
Mouse VICs differentiate to the osteoblast phenotype in response to calcifying medium using ascorbic acid, insulin, and phosphate stimulation. This article describes a quantitative method of calcium measurement using a kit and a qualitative method using Alizarin red staining. Both methods showed significant increase of calcification in response to calcifying medium treatment. The calcium measurement kit is the gold standard method, which offers an exact quantitative calcium measurement14.
In the Arsenazo III reagent, magnesium interference is prevented by the inclusion of 8-hydroxyquinoline sulfonate. Calcium reacts with the reagent to form a purple-colored complex, which absorbs at 650 nm. The intensity of the color is proportional to the calcium concentration. The accuracy of the Arsenazo-III reagent was previously validated with atomic absorption spectrophotometry. The same method is used in clinical laboratories to measure total calcium concentration in biological fluids14. The calcification in aortic stenosis is mainly hydroxyapatite, as shown with dispersive x-ray energy scanning electron microscopy analysis7,12,15. Indeed, it is important to analyze the calcification of the cell membrane rather than free calcium to more accurately mimic the calcification of the aortic valve tissue.
Mice represent a good source of VICs for the study of molecular mechanisms leading to aortic valve calcification. However, keep in mind that VICs in vitro are not similar to VICs in living valves. Another limitation is the fact that a pool of valves from 3-5 mice is needed to make a single cell culture. The pool should be from littermate mice to minimize variations. In addition, experiments should be performed in triplicate to confirm all findings. However, the use of the entire aortic valve in the culture can alleviate this limitation. Nevertheless, these in vitro studies must be validated in human tissue to strengthen the findings.
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 |