弁内皮細胞の単離

The isolation of resident interstitial and side specific endothelial cells from an aortic heart valve are demonstrated in this video protocol. The valve leaflet is harvested from the heart and placed in cold saline solution. The endothelial layer can be removed from both sides of the leaflet by gently shearing the tissue submerged in collagenase solution with a cotton swab for side specific isolation, the valve leaflet is pinned onto a paraffin bed so that the endothelial surface is exposed.

A few drops of cold collagenase are then added to the valve leaflet, and the leaflets are incubated for five to 10 minutes. After the incubation with collagenase, the side specific endothelial layer is removed by gently shearing of the tissue. With a swab, the released endothelial cells are then collected, washed and plated.

The remaining leaflets are then digested overnight to isolate the interstitial cells which are collected and washed. All the isolated cells are cultured and can be used to understand their role in heart valve repair and remodeling in response to local mechanical and biological signals. Hi, my name's Russell Gould.

I'm here at Cornell University in the biomedical engineering department working in the butcher lab. Today I'll be showing you how to isolate valvular endothelial cells. The the implications of this technique extend towards understanding how resident V cells constantly repair and remodel in response to mechanical and biological signals.

So let's get started. To begin, obtain a swine from your local abattoir and work aseptically to excise the aortic root. Thoroughly rinse the aortic root of blood.

With cold, sterile DPBS, it is imperative to remove all blood components as soon as possible to limit valvular endothelial cell or VEC, death and bacterial contamination. Antibiotics and antimycotic are not advised at this stage since they're potentially harmful to the ecs. Next, isolate directly from the root three.

Valve leaflets from each valve. Be conservative when isolating the leaflet as any annular tissue will contaminate the cell population. Place the isolated leaflets in a sterile 15 milliliter conical tube filled with 12 milliliters of cold DPBS.

Shake several times to remove debris and refill with fresh DPBS. Finally, transport the valve leaflets back to the lab on ice. Upon arrival at the laboratory place the container with the tissue under the sterile hood proceed to isolate the endothelial layer, autoclave the following in a covered instrument tray, serrated tissue forceps, tissue scissors, and cotton swabs.

Also prepare sterile collagenase solution endothelial medium and interstitial medium. As detailed in the accompanying written protocol. Fill a sterile 35 millimeter dish with three milliliters of cold collagenase solution per valve, and place all three leaflets from the 15 milliliter tube into the dish.

Incubate the tissue for five to 10 minutes at 37 degrees Celsius. Next, gently remove the endothelial layer from both sides of the leaflet by rotating a dry sterile swab onto the surface. The direction of rotation and the amount of shear applied are critical to the purity of the sample.

The rotation of the swab is in an opposite direction to the linear motion of the hand holding the cotton swab. This creates a controlled shear that lifts the endothelial cells from the tissue. The amount of force applied is enough to feel the resistance of the tissue, but without penetrating the basement membrane dab the swab occasionally within the collagenase solution to dislodge the cells from the tip fibers.

After swabbing is complete, the texture of the endothelial layer should feel slightly smoother than before. Collect the cells suspended in the collagenase solution and transfer to a new sterile 15 liter tube. After swabbing the endothelial cells off the leaflets, immediately place the leaflets in a 15 milliliter tube containing 10 milliliters of collagenase solution for interstitial cell isolation.

Centrifuge the tubes at 1000 RPM for five minutes at room temperature to pellet any isolated cells, aspirate the super natin. Add three milliliters of endothelial medium to the 15 milliliter tubes. Centrifuge a second time as before and aspirate the media.

This second centrifugation helps filter some of the unwanted materials such as tip fibers. Re-send the centrifuge endothelial cells in five milliliters of endothelial medium and plate the cells in a T 25 flask pre-coded with collagen. Using one flask per centrifuge tube, let the cells grow at least two to three days before changing the endothelial medium.

This helps the cells recover and divide. Since the isolation process is fairly harsh and the cell yield may be low, it's critical to passage cells near confluence. Since contact inhibition could lead to cell transformation, endothelial cells can also be isolated from a specific side.

To do so, first, prepare a Petri dish containing a paraffin bed line. 60 millimeter glass Petri dishes with aluminum foil. Calculate two glass dishes per leaflet.

The aluminum foil helps to remove the paraffin for reusing the petri dish. Next place paraffin beads in the dishes about half full and cover for autoclaving. Once autoclaving is complete and the paraffin is melted, move the dish ensemble to a cool flat surface.

As the paraffin cools, it hardens and creates a layer that will support needle punctures. After 60 minutes, the sterilized dish can be used to immobilize the leaflet tissue. Remove the leaflets isolated from the valve from the 15 milliliter tubes and place on the paraffin bed.

Manipulate the leaflets so that the ventricular side is face down on the paraffin surface, leaving the fibrosis side exposed. Pin the edges of the leaflet to expose the endothelial layer. Next place a few drops of cold collagenase on each upward facing endothelial surface and incubate the tissue for five to 10 minutes at 37 degrees Celsius.

When incubation is complete, gently remove the endothelial layer as before by rotating a dry sterile swab onto the surface of the leaflet. Occasionally dab the swab within the collagenase solution to dislodge cells from the tip fibers. After swabbing is complete, the texture of the endothelial layer should feel slightly smoother than before.

Collect the cell suspension and transfer to a new sterile 15 milliliter tube. Indicates side specificity on the label leaflets from the same valve can be pooled together. Rinse the immobilized valve using a few drops of cold DPBS to wash any remaining cells or debris.

Transfer the leaflets to a new culture dish so that the ventricular side is now exposed and collect endothelial cells as just shown. Once the swabbing of endothelial cells from the G leaflet is complete, immediately place the leaflets in a 15 milliliter tube holding 10 milliliters of collagenase solution. Wash the cells resus, suspend them and grow as shown for the non side specific isolation of the endothelial cells.

If the cell yield is very low, consider moving to a smaller flask or using a well plate. Since cell cell adhesion promotes cell growth, remember to passage the cells near confluence. Since contact inhibition could lead to cell transformation, start the interstitial cell isolation while centrifuging the endothelial cells incubate the tubes holding the leaflets for approximately 12 to 18 hours.

In collagenase solution, agitating gently if desired. At the end of the incubation period, use a serological pipette to mix the digestive tissue gently until the cell suspension becomes homogenized. This homogenization helps break up the tissue and release the interstitial cells centrifuge and wash the cells as shown for the endothelial cells.

Except use five milliliters of interstitial medium resus, suspend the centrifuge interstitial cells in 15 milliliters of interstitial medium and plate the cells in a T 75 flask Using one flask per centrifuge tube. Let the cells grow at least one to two days before changing the interstitial medium expect more tissue debris than with the endothelial cells. Also, the interstitial cells grow to confluence faster than the endothelial cells by nature and they provide higher cell yields in these representative images.

The morphology of cultured V Cs valvular endothelial cells and vs. Valvular interstitial cells are shown at two to three days. Becs exhibit a typical endothelial morphology in which the cells have a cobblestone pattern and begin to form clusters.

The VS.Exhibit morphology similar to myofibroblasts in which the cells are generally spindle shaped and sparsely populated when cultured to co fluency, ECS exhibit a typical endothelial morphology in which the cells of a cobblestone pattern and growth is contact inhibited. The VI CS exhibit morphology similar to myofibroblasts in which the cells are generally spindle shaped and not contact inhibited Functional characteristics of both VEC and VIC can be inferred from staining. For a ated LDL uptake.

VEC cells are associated with high levels of acetylated LDL uptake. While VIC cells are associated with low levels of acetylated LDL uptake, cell markers of VEC and VIC are shown here. ECS stain positive for von Willebrand factor while vs.

Do not exhibit von Willebrand factor staining. Furthermore, V CS stain negative for alpha smooth muscle acton while V CS stain positive for alpha smooth muscle actin. Well, I hope you learned a lot from our isolations today.

It's important to remember though that the endothelial cell basement membrane is very fragile and any disruption to the underlying matrix often liberates contaminating cells from the rest of the tissue. To confirm a successful isolation characterization is CRI critical to assess the purity of your isolations. So good luck.