Isolation of Mouse Valve Interstitial Cells: An Enzymatic Method to Isolate and Culture VICs from Mouse Aortic Valve

The heart's aortic valve is a tri-leaflet structure composed of the inner valve interstitial cells or VICs distributed throughout the dense extracellular matrix and the overlying valve endothelial cells or VECs.

To isolate VICs, first, prep a euthanized mouse in supine position. Through midline abdominal and chest incision, expose the heart. Make a small nick between the left atrium and ventricle followed by saline perfusion to drain the blood. Sever the ascending aorta at the aortic arch level and harvest the heart.

Transect and discard the distal two-thirds of ventricles before longitudinally dissecting the remaining portion of the heart to reveal the 'U-shaped' aortic valve leaflets. Excise the leaflets and rinse with chilled buffer to remove blood.

Subsequently, treat the leaflets with low concentrated collagenase solution and incubate. In low concentrations, collagenase - a proteolytic enzyme - only partially breaks down the extracellular matrix, dislodging surface VECs from the leaflets.

Centrifuge and replace the enzyme-containing supernatant with buffer, transferring the resuspended pellet into a Petri dish. Collect the leaflets and treat them with high concentrated collagenase solution for second digestion. Upon incubation, collagenase further degrades the extracellular matrix, releasing VICs into solution.

Centrifuge to pellet the VICs and resuspend in suitable media. Finally, seed a low volume of the cell suspension onto a multi-well plate to facilitate the adherence of VICs to the well bottom. Incubate to allow the VICs to proliferate.

Before starting the experiment, clean and sterilize all the surgical instruments and workspace with 70% ethanol and autoclave the surgical instruments for 30 minutes. When the initial setup is ready, start with cleaning the chest and the abdomen region of an 8-week-old euthanized mouse using ethanol.

Using scissors, open the abdomen and the chest of the mouse. Then, cut between the left atrium and the left ventricle with small surgical scissors. Remove blood from the heart by perfusing 10 milliliters of cold PBS and cut the heart keeping 3 millimeters of the ascending aorta. Under a stereomicroscope, dissect the aortic valve by cutting the heart horizontally in the middle of the ventricles and cutting the left ventricle towards the aorta. Then, carefully dissect the aortic valve. Pool the valves together in a small 35-millimeter tissue culture dish.

Wash the isolated valves in a 75-milliliter cell culture dish with 5 milliliters of freshly prepared 10 millimolar cold HEPES supplemented with antibiotics and incubate in 5 milliliters of collagenase type I for 30 minutes at 37 degrees Celsius with continuous shaking. After incubation, centrifuge the tube for 5 minutes at 150 x g and wash the pellet once with 2 milliliters of millimolar HEPES with vortexing at high speed for 30 seconds.

Collect the resultant suspension from the tube into a 35-millimeter culture dish and use thin tweezers to carefully transfer the tissue fragments into a fresh tube. Then, incubate the pellet in a 15-milliliter tube at 5 milliliters of collagenase type I at 37 degrees Celsius under continuous agitation for 35 minutes. Separate the cells by resuspending with a 1-milliliter pipette and centrifuge at 150 x g for 5 minutes at 4 degrees Celsius. Clean the cells twice by resuspending the separated pellet in 2 milliliters of complete DMEM and centrifuging at 150 x g for 5 minutes at 4 degrees Celsius.

For plating, resuspend the pellet in 1 milliliter of complete medium and add it to one well of a 6-well cell culture dish in a minimum amount of medium. Keep the plates undisturbed at 37 degrees Celsius with 5% carbon dioxide. After 3 days of incubation, confirm growth closer to tissue debris by observing the cells under the microscope. Once 1,000 cells are visible, carefully remove the tissue debris with autoclaved tweezers and change the medium.