Isolation of Murine Valve Endothelial Cells

The overall goal of this procedure is to isolate enriched populations of heart valve, endothelial cells from embryos and adult mice with an endothelial reporter. This is first accomplished by careful dissection of the atrial ventricular canal region containing all mitral, tricuspid, aortic, and pulmonic heart valve leaflets. The second step of the procedure is to dissociate the valve tissue and release individual cells by treating the tissue to a series of enzymatic treatments.

This process is repeated nine times. Then the solution containing cells and debris is passed through a filter to ensure a single cell suspension without cell debris. The final step is to sort and collect the dissociated cells using fluorescence activated cell sorting.

Ultimately, a sufficiently large population of enriched valve endothelial cells is made available for in vitro culturing and molecular analysis. In this approach, we will show how to isolate heart valve endothelial cells from mice. As biomolecular tools have been well established in the mouse model.

This allows for an expanded set of experimental designs to be used for heart valve research. Additionally, isolation of heart valve endothelial cells in embryos allows for previously unfeasible developmental studies to be performed. Let's get started.

Be sure to completely sterilize the surgical tools by autoclaving them on an instrument tray. The surgical site should be sanitized by spraying it down with 70%ethanol. This includes the microscope and any other potentially contacted equipment at the site.

The following solutions must all be prepared immediately prior to the experiment. Dissociation buffer, sorting buffer, and if culturing, VEC culture, media and GFP negative culture media. Each of these solutions must be sterilized with a 0.2 micron filter, after which the solutions must be kept on ice until they are used after carbon dioxide.

Asphyxiation of the mouse, followed by cervical dislocation. Use dissection scissors to open the chest cavity. The mice here are homozygous for tie two GFP or are age matched negative controls required for fact sorting.

Next, expose the heart and lungs. Then grip the heart with forceps at the great arteries, and pull it away from the body. Partially submerge the heart.

In a dish of cold HBSS in the dish, prepare the heart for dissection by removing the attached lung tissue, it is critical to eliminate non-valvular endothelial cells that could contaminate the sample. To obtain an atrial ventricular canal ring first, remove the atria by gently pulling them away from the heart. Next, use a razor blade to cut the ventricles off, making the cut right below the AV valves, the aortic regions should remain intact.

Next, make an incision down one side of the canal to open the ring and expose the atrial ventricular valve structures using forceps. Gently tease away the myocardium to expose the valve leaflets. They are dense white tissue over the myocardium.

Now, gently detached the cordi tendai from the atrial ventricular valves if it is still attached. And remove the proximal regions of the pulmonary artery and aortic walls that do not contain the semilunar valve structures in the process. Remove as much of the remaining myocardium as possible without damaging the valve leaflets as the ECS may get dislodged.

A careful dissection is critical to eliminate non-valvular endothelial cells that could contaminate the sample. So try to remove as much of the surrounding tissue as possible while keeping all four valves intact. Finally, put the trimmed valvular heart regions into a 1.5 milliliter tube with one milliliter of cold HBSS and keep the tube on ice.

Use this technique to prepare isolated valvular tissue from adult or E 14.5 animals with a pipette. Remove the HBSS from the tube with the tissue. Then add back a milliliter of dissociation buffer and four microliters of DNAS one.

Incubate the tissues in the dissociation buffer and DN with gentle rotations at 37 degrees Celsius for seven minutes. After seven minutes, pipette the tissues up and down three times to help dissociate the cells. After settling for about 15 seconds, collect the supernatant containing the dissociated cells.

Transfer the cells to a 15 milliliter conical tube. End the collagenase reaction in the 15 milliliter conical with an addition of horse serum. This is the first collection fraction.

Now returning to the tube of the settled tissues, add back dissociation buffer and DNAS one. Repeat the procedure to collect a second fraction. Keep repeating the process until a total of nine cell suspensions are collected.

Now pass all the fractioned collections through a 70 micron nylon filter into a single tube for a debris free suspension of cells. Collect the cells into a pellet by spinning the tube down at 400 GS for five minutes. Then resuspend the pellet into a milliliter of sorting buffer and keep them on ice until they can be analyzed by fax, which is covered in the text.

Protocol cell culture and staining are also covered by the text protocol tissue sections from tie two GFP transgenic. Mice were collected from adults and E 14.5 embryos in adults. The valve endothelial cells were identified with the endothelial marker CD 31 and were found to coex express GFP.

The same cells isolated from E 14.5 embryos. Also co-expressed CD 31 and GFP fax analysis identified GFP positive and negative cell populations from cells prepared from adult and E 14.5 embryos. Wild type C 57 black six.

Adult cells were used to set the GFP parameters gene expression in the GFP positive and GFP negative cell populations were compared by quantitative PCR in the GFP positive cells. Endothelial markers were high and myocyte markers were very low. In GFP negative cells.

Valve interstitial cell markers were high. The two cell populations were cultured separately after two weeks. The GFP positive cells took on around morphology and expressed CD 31.

By contrast, the GFP negative cells took a mesenchymal like morphology after nine days. These cells also expressed the VIC marker alpha SMA After its development. This technique paved the way for researchers in the field of heart valve biology to be able to explore mechanisms of development and disease in mouse models that can then be translated to the human population.