Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

The overall goal of this procedure is to culture mouse cardiac valves using the miniature tissue culture system or MTCS. This is accomplished by first assembling the MTCS without the profusion chamber and circulating the medium to precondition the system. In the second step, the mouse heart is harvested and prepared for cannulation.

Next, the heart is placed in the profusion chamber where it is cannulated for a culture of the mitral or aortic valve. The perfusion chamber is then placed in the MTCS and the pump has started. Ultimately, histological analysis is used to assess the viability, morphology and extracellular matrix distribution of the valve.

Begin by using pieces of silicone tubing to make connections between the reservoir and the pump. The pump and the bubble trap, and the bubble trap and the reservoir. When all of the tubing is connected, fill the reservoir with 70%ethanol.

Turn the pump on at one milliliter per minute, flow speed, and let the ethanol circulate to sterilize the tubing. After 30 minutes, empty the reservoir and pump the ethanol out of the system. Next, fill the reservoir with sterile distilled water and pump the water through the system to remove the residual alcohol.

Then pump the water out again and fill the reservoir with 45 milliliters of freshly prepared medium. Circulate the medium to fill all of the tubing and allow the medium to adapt to the gas composition in the incubator for at least one hour. Under standard tissue culture conditions.

To isolate the mouse heart, first, inject the animal with 500 units of heparin ip. After 10 minutes, place the mouse on a dissection board and confirm the appropriate level of anesthesia by toe. Pinch, sterilize the fur with 70%ethanol, and then use a pair of scissors to open the abdominal cavity, exposing the venina cava and diaphragm to visualize the heart, make lateral incisions starting from the bottom and ending at the top of the ribs.

Then reflect the thoracic cage over the animal's head and observe the heart beating. Next, insert a 21 gauge needle attached to a five milliliter syringe containing sterile tyros buffer through the diaphragm into the inferior vena cva from the abdominal into the thoracic cavity. Make sure that the blood can exit from the vena cva, coddle from the needle insertion, and then perfused the buffer gently and with constant pressure into the vena cava until the heart partly loses its red color.

Now switch out the syringe of buffer for five milliliters of sterile potassium chloride solution, and gently perfuse the solution into the vena CVA until the heart stops beating. Using curved forceps and scissors, slightly lift and dissect the heart from the surrounding tissue. Then keep the organ in a 10 milliliter tube containing ice cold PBS supplemented with antibiotics and anti mycotic for up to three hours to cannulate the heart, transfer it into a 10 centimeter Petri dish within a laminar flow hood, and add fresh PBS supplemented with antibiotics and antimycotic Using a dissecting microscope, micro scissors and forceps, remove all of the non-cardiac tissue, preserving the ascending aorta to at least the bifurcation with the brachiocephalic artery and the pulmonary veins, two millimeters proximal to the heart.

Then cut two millimeters off of the tip of the apex to create access to the left ventricular lumen and placed a perfusion chamber in the hood using pieces of silicone tubing. Next, attach five milliliter syringes filled with medium to the number one needles for culture of the mitral valve, and number two, needles for culture of the aortic valve. And fill the profusion chamber with 20 milliliters of medium.

Then put the heart on the rotation stage in the perfusion chamber between the two blunted needles, and adjust the height of the stage so that the heart is positioned in front of the needles to culture. The mitral valve ligate the aorta and the left atrial appendage with a silk 7.0 suture. Then insert the needle number one through the pulmonary vein into the left atrium, and place a suture proximal to its entry in the left atrium.

Taking care that the needle does not enter too far into the left atrium, insert the needle number two into the left ventricle. Then transfer the medium from the profusion chamber into a 50 milliliter tube, and use biocompatible glue to seal the needle to the myocardium. When the glue is dry, use the needle number one syringe to carefully inject the medium into the heart.

Check the heart for leakage and confirm that the medium exits from the needle. Number two, and that the remaining blood is perfused out of the tissue. To culture the aortic valve, insert the needle number one into the aorta and ligate it with a suture as just demonstrated taking care not to place the needle too far into the aorta.

Then insert the needle number two, into the left ventricle, seal the needle and inject the medium as just demonstrated. If there is no leakage, fill the profusion chamber with 20 milliliters of the transferred medium. Place the gasket and the lid on the chamber and tighten both with a washer and screws.

Place the profusion chamber onto the stand. Finally, place the profusion chamber into the stand and start the pump. With a flow speed of around 600 microliters per minute, the aortic and mitral valves can be cod for at least three days with no cell death observed during the culture as determined by the absence of tunnel positive cells.

Further, the collagen distribution as visualized by Massen Trichome staining is similar to that observed for the native condition. When cultured in 1%serum, the valves are responsive to changing culture conditions. For example, as seen here, increasing the amount of serum to 10%results in thicker leaflets.

Furthermore, a clear collagen free region is observed at the ventricular side of the leaflet near the attachment to the aortic wall. Together these observations demonstrate that the cultured valves remain viable, resembled their native counterparts, and are responsive to environmental changes after its development. This technique paved the way for researchers in the field of valvular biology to explore the mechanisms that regulate valvular homeostasis and disease.