Isolation and Culture of Aortic Smooth Muscle Cells and In Vitro Calcification Assay

Fig. 1

Double-collagenase digestion procedure for VSMC isolation from aorta

3.2 Mouse Aorta Dissection

Select mice from which aortas will be dissected. Usually 5–10 mice are required for one VSMC isolation procedure (see Note 4 ). The same protocol can be used for rat aortas.

Euthanize the first mouse (see Note 5 ).

Spray the mouse with 70 % of ethanol and make an initial cut in the lower part of the abdomen.

Cut the skin from both sides to visualize the organs and the rib cage.

Open the chest cavity by cutting the ribs from both sides to access the heart.

Remove or move organs (liver, spleen, intestine, etc.) to reach the aorta, which is visible as a white tube running along the spine.

With scissors, cut the abdominal aorta below the renal arteries.

With a syringe, inject 5 mL of PBS into the left ventricle in order to flush blood out of the aortic lumen.

Carefully dissect the aorta away from the vertebral column using small surgical scissors (straight or curved) and forceps (see Note 6 ). Start above the abdominal part of the aorta where the diaphragm is attached and work towards the heart.

10.

Separate the aorta from the heart; make a cut before the aortic arch to obtain only the thoracic part (see Note 7 ). Put the isolated aorta in a culture dish containing PBS (see Fig. 2a).

Fig. 2

Cleaning mouse aorta. (a) Aorta freshly dissected from a mouse. (b) Aorta after removal of the surrounding tissue. (c) Removal of the adventitia (held with left tweezers) from the medial layer (held with right tweezers)

11.

Follow the same procedure (steps 2–7) for all mice. Store all the dissected aortas in PBS until further use (next Subheading 3.3).

3.3 Vascular Smooth Muscle Cell Isolation and Culture

Use a microscope and two microdissection tweezers to remove the tissue surrounding the newly isolated aortas (from Subheading 3.1) and obtain clean aortas (see Fig. 2b).

Transfer clean aortas into a culture dish containing fresh PBS.

Put all aortas in a 35 mm dish containing 5 mL of the collagenase solution (Subheading 3.1, step 2). Incubate for 15 min at 37 °C in 5 % CO₂ without agitation (see Note 8 ).

Put one aorta in a culture dish with fresh PBS.

Viewing the aorta under a microscope, use two microdissection tweezers to remove the adventitia (see Fig. 2c). The adventitia should be rolled off the medial aorta in a manner similar to removing a sock from a foot (see Note 9 ).

Repeat steps 4 and 5 for all the aortas.

Place the medial aortas in a sterile culture dish containing sterile PBS using a sterile scalpel blade. From this step work in STERILE conditions!

Cut each aorta into small fragments using a blade (see Note 10 ).

To release cells from the aortic fragments, place these in a 50 mL sterile tube containing the remaining collagenase solution (Subheading 3.1, step 2) and a magnetic stir bar and incubate at 37 °C for 90 min (see Note 11 ) on a magnetic stirrer with gentle mixing (see Note 12 ).

10.

At the end of the digestion period, remove the stir bar with sterile forceps. Centrifuge the cell/collagenase mixture for 5 min at 300 × g. Remove the supernatant and resuspend the cell pellet in 50 mL of sMEM.

11.

Centrifuge the suspended cells for 5 min at 300 × g. Remove the supernatant and resuspend the cell pellet in an appropriate volume of sMEM supplemented with 10 % FBS (see Table 1).

Table 1

Growth area and medium volume for selected cell culture vessels

	Approx. growth area (cm²)	Recommended medium volume (mL)
Multiple-well plates
6 well	9.5	1.9
12 well	3.8	0.8
24 well	1.9	0.4
48 well	0.95	0.2
Dishes
35 mm	9	1.8
60 mm	21	4.2
100 mm	55	11
Flasks
25 cm²	25	5
75 cm²	75	15

12.

Place the cells in two wells of a 12-well plate (see Note 13 ). Under the microscope, confirm the presence of round cells in suspension (see Note 14 ). Incubate at 37 °C in 5 % CO₂ atmosphere.

13.

After 2 days, remove the sMEM (with debris) and add fresh sMEM supplemented with 10 % FBS. Under the microscope, confirm the presence of adherent cells (see Fig. 3a).

Fig. 3

Primary culture of vascular smooth muscle cells (VSMCs). VSMCs were incubated at 37 °C in 5 % CO₂ with MEM supplemented with 1 mM l-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 10 % FBS. (a) Groups of VSMCs after 2 days of culture. (b) A group of adherent VSMCs after 1 week of culture. (c) VSMCs replated after the first trypsinization passage

14.

Change the medium every 2 days (use sMEM supplemented with 10 % FBS), each time observing cells under the microscope to monitor cell growth.

15.

When numerous cell colonies are formed (see Fig. 3b), wash cells twice with PBS and add 0.5 mL of 1× trypsin per well (see Note 15 ); incubate for ~5 min at 37 °C in 5 % CO₂ until cells detach.

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