成体マウスの心筋細胞の単離、培養および形質導入

The overall goal of this procedure is to isolate viable primary adult mouse cardiomyocytes for culture and adenoviral vector transduction. This method will answer some the key question in the field of cardiovascular research. In particular, we can adjust what prevents adult mammalian cardiomyocytes from cell cycle entry and completion.

The main advantage of this technique is that a high yield of viable adult mouse cardiomyocytes can be obtained for long term culture. Visual demonstration of this method is critical, as the heart extraction and cannulation procedures are difficult, but must be performed quickly and efficiently. To extract the heart, begin by treating an adult mouse with five international units of heparin per gram of body weight by intraperitoneal injection.

Wait 20 minutes to allow the heparin to circulate to the heart. Then, after administering anesthesia, confirm a lack of response to toe pinch and corneal reflexes and tape the animal's limbs to a surgery platform. Sterilize the incision area, with liberal application of 70 percent ethynol, and then pat the hair dry with a lab wipe.

Next, use forceps to lift the skin just below the sternum and make a lateral incision through the skin and peritoneum. Then, using hemostats, gently lift the rib cage via the sternum and use small dissection scissors to extend the incision in a V shape toward the axially. Cut through the first rib and puncture the diaphragm on each side, keeping the rib cage lifted with the hemostats.

Using small Iris scissors, completely transect the diaphragm. Then use the hemostats to retract the ribcage rostrally and quickly but carefully extend the incision through the ribcage toward the axially on both sides. Retract the middle section of the ribcage fully, laying down the attached hemostats to hold the tissue in place, and remove the pericardium with fine forceps.

Next, use curved forceps to gently lift the heart and transect the attached veins and arteries. Wash the heart in ice cold perfusion buffer, then transfer the heart onto a small piece of 215 micron mesh. Add ice cold perfusion buffer to slow myocardial contractions, and place the dish under a dissection microscope.

Using fine iris scissors, trim any access non-cardiac tissue. Then transect the aorta near the brachiocephalic artery, taking care not to let bubbles or tissue debris enter the opening. To cannulate the heart, fill the Petri dish with ice cold perfusion buffer until the solution rises above the opening of the 18 gauge cannula.

Then, keeping the tip of the cannula and aorta below the surface of the buffer, sheath the aorta onto the cannula until the needle tip is just distal to the aortic valve. Slide a pre-tied 7-0 silk suture down the shaft of the cannulation needle until the suture is just above the needle tip and tighten the knot with fine forceps. Place a second suture just above the first.

Then slowly depress the syringe until 0.5 milliliters of perfusion buffer has been injected through the coronary vasculature. After removing the syringe, attach the cannula to the perfusion outlet port. At the end of the perfusion, use forceps to remove the heart from the cannula, and place the heart in an empty Petri dish.

Using fine Iris scissors, quickly trim the extra ventricular tissue and wash the ventricles in a new Petri dish containing perfusion buffer. After washing, transfer the ventricles into a dry Petri dish and place in a sterile hood. Collect 7.5 milliliters of digestion buffer from the heat jacket of the perfusion system, and add 2.8 microliters of one molar calcium chloride.

Mix the solution by inversion. And then add two to three milliliters of the calcium chloride supplemented digestion buffer to the ventricles. Using fine forceps, quickly tritcherate the ventricular tissue until most of the pieces are smaller than one cubic millimeter, and add the remaining calcium chloride supplemented digestion buffer to the dissociated tissues.

Mix the fragments by gentle agitation. And incubate them at 37 degrees Celsius and five percent carbon dioxide with continued agitation every two minutes until a sufficient number of cardiomyocytes have been released. Then return the dish to the laminar flow hood and use a transfer pipette to gently dissociate the tissue further.

Wearing sterile gloves, fold the 215 micron mesh into a cone and hold it over a 50 milliliter conical tube. Pipette the tissue slurry onto the mesh, and allow the filtrate to drain into the tube. Followed by a wash with 7.5 milliliters of pre-warmed stop buffer.

Transfer the cell suspension to a 15 milliliter conical and allow the cells to settle by gravity for 15 minutes. Then use a new transfer pipette to carefully remove the supernatant until only 50 to 100 microliters of solution remains above the cells. Add 10 milliliters of pre-warmed equilibrated culture medium to the cells, and mix by gentle inversion.

Finally, after another 15 minute room temperature incubation, use a new transfer pipette to remove the supernatant from the settled cells, until only 50 to 100 microliters of medium remain. Immediately after isolation, the cardiomyocytes maintain a mostly rod-shaped appearance with intact sarcomeres and a sharp outer membrane under bright field illumination. Immunostaining for cardiomyocyte-specific sarcomeric markers such as alpha actinin reveals a distinct sarcomeric banding pattern.

After several days in culture, the cardiomyocytes assume a rounded morphology. Within one to two weeks, the live cardiomyocytes attach to the substrate and begin spreading. Transduction with adenovirus, as observed in these GFP positive cardiomyocytes, can be used to achieve a strong gene expression within 48 to 72 hours.

Once mastered, this technique can be completed in four hours if it is performed properly. While attempting this procedure, it is important to remember to achieve a secure cannulation that is free of air bubbles in order to efficiently profuse the coronary vasculature. Following this procedure, other methods such as immunostaining or live imaging can be performed in order to answer questions such as why are adult mammalian cardiomyocytes resistant to cell division?

Don't forget that working with adenovirus can be extremely hazardous, so appropriate precautions must be followed, such as working in a class two biosafety cabinet.