The use of isolated mouse valve cells is essential to investigate the signaling pathway leading to valve calcification in the use of genetically modified cells from transgenic mice. The two-step digestion is a quick and efficient method. The most challenging part of this protocol is the isolation of the aortic valve from eight weeks mice.
It is better to practice on older mice to visualize the aortic valve better. 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 eight 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 three 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 five milliliters of freshly prepared 10 millimolar cold HEPES supplemented with antibiotics and incubate in five milliliters of collagenase type one for 30 minutes at 37 degrees Celsius with continuous shaking. After incubation, centrifuge the tube for five minutes, at 150 times G and wash the pellet once with two milliliters of 10 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 with five milliliters of collagenase type one at 37 degrees Celsius under continuous agitation for 35 minutes. Separate the cells by resuspending with a one milliliter pipette and centrifuge at 150 times G for five minutes at four degrees Celsius.
Clean the cells twice by resuspending the separated pellet in two milliliters of complete DMEM and centrifuging at 150 times G for five minutes at four degrees Celsius. For plating, resuspend the pellet in one milliliter of complete medium and add it to one well of a six well cell culture dish in a minimum amount of medium. Keep the plates undisturbed at 37 degrees Celsius with 5%carbon dioxide.
After three days of incubation, confirm growth close to the 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. Trypsinize the cells after reaching 70%confluency and transfer them to a 75 milliliter tissue culture dish.
Before beginning the assay, clean the biosafety hood with 70%ethanol and warm the DMEM medium to 37 degrees Celsius. Next, seed 100, 000 cells per condition into six well plates in complete DMEM and culture at 37 degrees Celsius. After 24 hours, replace the supernatant medium with the calcifying medium and incubate the cells for seven days at 37 degrees Celsius, replacing the medium on the third day.
After seven days, measure the calcium concentration in a 96 well plate by using the Arsenazo III reagent and recording the absorbance at 650 nanometers. In the representative analysis, various valve cell phenotypes were identified with immunofluorescent staining after three to five days of culture. Mouse VIC's expressed vimentin and alpha SMA, the significant markers of valve cells.
Additionally, CD31 immunofluorescent staining verified no contamination from endothelial cells in VIC culture. Culturing VIC's with phosphate rich calcifying medium stimulate a calcification in cells, further confirmed with the red positive staining for calcium nodes. The protocol is based on a pool of three to five valve from different mice.
The use of litter mate and three different biological replicates is important to validate the findings. The use of mouse valve cells is vital to understand the molecular pathway leading to aortic stenosis by isolating cells from transgenic mice. This protocol has been used previously to investigate the implication of pathway to receptor in mineral regression of calcified aortic valve.
