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